IRLF 


The  Ruling  of  the  Turck  Disc.  Explanation  of  the  Squares. — In  the  first  place, 
we  have  the  square  which  encloses  the  entire  ruled  surface.  This  is  made  up  of 
nine  squares,  each  i  mm.  square.  These  are  the  squares  to  use  in  connection  with 
leukocyte  counts  with  the  white  pipette.  They  may  be  termed  the  large  squares. 
The  very  smallest  squares  which  can  be  found  are  those  made  by  the  intersection  of 
the  triple  ruled  lines  in  the  center;  they  are  1/40  mm.  or  25  microns  square  and  are 
never  used  for  any  purpose,  except  possibly  in  connection  with  the  counting  of  bacteria 
in  a  vaccine.  It  will  be  observed  that  it  requires  four  of  these  very  small  squares  to 
make  one  of  the  squares  usually  designated  as  the  small  square. 

There  are  400  small  squares  in  each  large  square,  consequently,  as  there  are  nine 
large  squares,  the  entire  ruled  surface  consists  of  3600  small  squares.  There  are 
4000  small  squares,  in  i  cubic  mrn. 

The  unit  in  estimating  the  leukocyte  or  red  cell  content  of  blood  is  the  cubic 
millimeter.  The  unit  is  i/iooo  of  a  cubic  centimeter. 

In  making  a  leukocyte  count  we  usually  take  the  white  pipette,  which  has  the 
mark  II  just  above  the  bulb,  and  draw  up  the  blood  to  0.5  and  then  with  suction  we 
fill  the  pipette  to  the  II  mark  with  the  diluting  fluid  for  which  a  1/2%  solution  of 
glacial  acetic  acid  in  water  is  most  satisfactory.  This  gives  a  dilution  of  1-20. 

Counting  with  the  2/3  inch  objective  all  of  the  highly  refractile  dots  representing 
leukocytes  in  one  of  the  i  mm.  squares  at  either  of  the  four  corners  we  note  the  num- 
ber and  mentally  multiply  by  20  (the  number  of  times  the  blood  was  diluted).  As 
the  depth  of  the  diluted  blood  between  the  ruled  surface  of  the  haemacytometer 
slide  and  the  under  surface  of  the  cover-glass  is  only  i/io  of  a  millimeter,  we  multiply 
the  figure  as  above  obtained  by  10  to  get  the  number  of  cells  in  a  1-20  dilution  of 
blood  in  a  space  of  one  cubic  millimeter. 

Example:  Counted  90  leukocytes;  90X20=1800X10=18,000:  equals  number 
of  leukocytes  in  i  cubic  mm.  of  blood. 

For  red  counts  we  use  the  red  count  pipette  which  has  the  101  mark  just  above  the 
bulb.  Taking  up  blood  to  0.5  we  draw  up  the  diluting  fluid  to  101.  This  gives  a 
dilution  of  1-200.  Counting  the  red  cells  in  five  of  the  aggregations  of  16  small 
squares  (1/20  mm.)  thus  having  counted  80  small  squares  we  have  counted  1/50 
of  the  total  number  of  small  squares  in  a  cubic  mm.,  there  being  4000  small  squares 
in  a  cubic  mm.  Consequently  the  number  of  red  cells  in  80  small  squares  multiplied 
by  50  and  then  by  the  dilution  of  200  gives  the  number  of  red  cells  in  one  cubic 
mm.  of  the  blood  examined. 

It  is  well  to  make  a  second  preparation  and  record  the  average  of  the  two  counts. 


OR,  HENKY  HORH 


VST  &  GRANT  AVE, 

SAA'  FRANCISCO,  CAL 


Principal  normal  and  pathological  blood-cells  with  average  size, 
percentage  in  a  normal  differential  count  and  the  diseases  in  which 
certain  pathological  cells  are  more  or  less  pathognomonic. 


LIBRARY  OF 

r  :FORNIA 


The  diameter  of  the  bottom  of  this  Petri  dish  is  3  inches  or  7.5  + 
centimeters. 

The  area  of  a  circle  is  equal  to  the  square  of  the  radius  multiplied 
by  TT  or  22/7. 

i  1/2  in.=radius.  i  1/2X1  1/2  =  2.25.  2.25X22/7  =  7.07  square 
inches. 

3.75  cm.  =  radius.  3.75X3.75  =  14.06.  14.06X22/7=44.1  square 
centimeters. 

Number  of  bacterial  colonies  in  i  sq.  in.  averages,  approximately, 
75.  Number  in  7.07  sq.  in.  =  530. 

Number  of  bacterial  colonies  in  i  sq.  cm.  averages,  approximately, 
12.  Number  in  44.1  sq.  cm.  =  5 28. 

In  a  microscopic  field,  if  the  diameter  were  8  small  squares  (1/20 
mm.),  the  radius  would  be  4  small  squares  and  the  area  of  such  a  round 
field  would  be  4X4  =  16X22/7  =  50+.  Such  a  field  would  contain  50 
small  squares. 


PRACTICAL  BACTERIOLOGY,  BLOOD  WORK 
AND  ANIMAL  PARASITOLOGY 

STI  TT 


BY  THE  SAME  AUTHOR 


The  Diagnostics  and  Treatment 


OF 


Tropical    Diseases 

ILLUSTRATED  PREPARING 


PRACTICAL 

Bacteriology,  Blood  Work 

AND 

Animal  Parasitology 

INCLUDING 

Bacteriological    Keys,    Zoological    Tables 
and  Explanatory  Clinical  Notes 


BY 

E.  R.  STITT,  A.  B.,  Ph.  G.,  M.  D. 

MEDICAL   INSPECTOR,   U.   S.    NAVY;   GRADUATE,   LONDON    SCHOOL     OF  TROPICAL   MEDICINE;    HEAD 
OF  DEPARTMENT   OF  TROPICAL  MEDICINE,   U.    S.    NAVAL  MEDICAL  SCHOOL;  PROFESSOR  OF 
TROPICAL  MEDICINE,   GEORGETOWN   UNIVERSITY;    LECTURER    IN    TROPICAL    MEDI- 
CINE,  JEFFERSON   MEDICAL  COLLEGE;   FORMERLY     ASSOCIATE   PROFESSOR 
OF   MEDICAL  ZOOLOGY,    UNIVERSITY  OF  THE  PHILIPPINES   AND 
INSTRUCTOR     IN    BACTERIOLOGY     AND     PATHOLOGY, 
U.     S.     NAVAL     MEDICAL     SCHOOL. 


Third  Edition,  Revised  and  Enlarged 
With  4  Plates  and  106  Other  Illustrations  Containing  513  Figures 


PHILADELPHIA 

P.   BLAKISTON'S   SON   &   CO/ 

1012   WALNUT   STREET 
1914 


FIRST   EDITION,    COPYRIGHT,    1909,   BY   P-    BLAKISTON'S   SON   &    Co. 

SECOND    EDITION,     COPYRIGHT,     1910,    BY    P.    BLAKISTON'S    SON    &    Co. 

THIRD   EDITION,    COPYRIGHT,    1913,   BY   P.   BLAKISTON'S   SON  &    Co. 


?T,H  %  .  yrAf&Lp  .PRESS. YORK. PA 


01 


PREFACE  TO  THE  THIRD  EDITION. 


In  the  preparation  of  the  third  edition  of  this  laboratory  manual  it 
soon  became  evident  that  the  new  material  to  be  added  would  increase 
the  size  of  the  book  beyond  that  which  would  permit  its  being  readily 
carried  in  one's  pocket.  It  has,  however,  been  possible  to  keep  the  size 
of  the  book  within  the  limits  considered  desirable  by  the  use  of  a  smaller 
type  in  a  considerable  proportion  of  the  paragraphs  so  that  in  this  way 
and  by  increasing  the  number  of  lines  on  each  page  it  has  been  possible 
to  add  extensively  to  the  subject  matter  and  with  only  an  increase  of 
about  sixty-five  pages. 

The  advantage  attaching  to  more  ready  reference  obtained  by  the 
alternation  of  different  sizes  of  type  would  appear  to  make  this  plan  an 
improvement  over  the  old. 

While  the  chapters  dealing  with  bacteriology  have  been  added  to 
and  made  to  include  more  recent  advances  it  will  be  noted  that  in  the 
section  on  animal  parasitology  the  subject  matter  has  been  greatly 
increased. 

In  the  revision  of  the  chapter  on  protozoa  I  am  greatly  indebted  to 
Professor  Minchin's  recent  work  on  the  Protozoa  and  in  those  relating 
to  arachnoids  and  insects  to  the  very  practical  volume  of  Colonel 
Alcock  entitled  "  Entomology  for  Medical  Officers." 

The  illustrations  have  been  added  to  and  many  which  did  not  seem 
to  bring  out  sufficiently  details  of  anatomy  have  been  replaced  by  others 
more  satisfactory  in  that  respect. 

The  three  plates  of  the  cestode,  trematode  and  nematode  ova  were 
drawn  by  Mr.  L.  Avery  under  the  supervision  of  P.  A.  Surgeon  Garrison, 
U.  S.  N.  and  it  is  believed  that  they  will  be  found  more  satisfactory  than 
similar  plates  contained  in  works  on  animal  parasitology. 

Several  new  tables  have  been  added  among  which  special  attention 
is  called  to  the  one  on  urinary  findings  in  various  diseases  of  the  genito- 
urinary system  and  also  to  the  key  to  the  intestinal  bacteria  attached 
to  the  inside  of  the  board  cover. 

A  chapter  on  " Disinfectants  and  Insecticides"  giving  the  practical 


VI  PREFACE    TO    THE    THIRD    EDITION 

application  of  methods  of  carrying  out  these  important  Public  Health 
questions  has  been  added. 

In  the  chapter  on  " Immunity"  a  modification  of  Emery's  technic 
for  the  Wassermann  test  has  been  incorporated — the  use  of  Noguchi's 
reagents  with  Emery's  technic.  The  subject  matter  of  the  sections  on 
vaccines  and  anaphylaxis  has  been  extensively  revised. 

E.  R.  S. 


PREFACE  TO  THE  SECOND  EDITION. 


THE  fact  of  the  necessity  for  a  second  edition  of  this  manual  of  lab- 
oratory and  clinical  diagnosis  in  a  little  more  than  a  year  would  indicate 
that  the  original  arrangement  of  material  should  be  adhered  to. 

Each  section  of  the  book  had  been  carefully  revised  and  much  new 
matter  added.  In  particular  has  that  part  of  the  book  relating  to  ani- 
mal parasitology  been  rewritten  and  almost  doubled  in  extent,  and  a 
chapter  on  Poisonous  Snakes  added.  In  the  chapter  on  "  Practical 
Methods  in  Immunity"  the  most  recent  advances  in  the  Wassermann 
test  and  practical  agglutination  methods  have  been  incorporated  as  well 
as  a  brief  discussion  of  the  question  of  Anaphylaxis. 

The  section  on  "  Clinical  Bacteriology  and  Animal  Parasitology  of 
the  Various  Body  Fluids  and  Organs"  has  been  revised  to  meet  the  most 
recent  advances  in  clinical  diagnosis.  This  section  not  only  answers 
as  a  cross  index  to  the  importance  of  the  various  bacteria  and  animal 
parasites  in  practical  clinical  work,  but  gives  a  concise,  practical  state- 
ment as  to  how  to  proceed  in  the  examination  of  various  secretions  and 
excretions.  This  information  is  difficult  to  obtain  in  the  larger  works 
on  clinical  diagnosis  by  reason  of  its  being  taken  up  under  many  different 
headings. 

A  method  is  given  for  the  making  of  differential  counts  in  the  same 
preparation  as  that  for  making  the  leukocyte  count  which  has  the  advan- 
tages of  accuracy  and  the  saving  of  time. 

Several  new  illustrations  have  been  added — the  one  of  poisonous 
snakes  has  been  taken  from  Stejneger's  report. 

The  plan  of  making  this  little  volume  a  practical  one  has  been  con- 
tinued in  the  second  edition;  theoretical  considerations  have  been 
brought  out  only  when  necessary  to  a  proper  understanding  of  some 
recent  or  difficult  laboratory  method. 

The  very  elementary  considerations  and  definitions  have  not  been 
given  because  in  order  to  present  a  compact  and  at  the  same  time  a 
practical  working  guide  it  has  been  necessary  to  eliminate  that  which 

vii 


viii  PREFACE    TO    THE    SECOND    EDITION 

seemed  least  essential.  Furthermore,  instruction  in  biological  science 
is  now  a  part  of  the  requirements  of  candidates  for  admission  to  the 
various  medical  schools. 

At  the  request  of  many  who  have  found  the  book  of  assistance  I 
have  added  an  outline  of  those  methods  in  the  chemical  examination 
of  urine  and  gastric  contents  which  have  seemed  to  me  to  be  most  essen- 
tial in  the  making  of  diagnoses.  In  the  tropics  I  have  found  the  deter- 
minations of  total  nitrogen  and  nitrogen  eliminated  as  ammonia  to  be 
exceedingly  valuable  in  diagnosis.  Methods  for  such  determinations, 
as  elaborated  by  Assistant  Surgeon  E.  W.  Brown,  U.  S.  Navy,  of  the 
U.  S.  Naval  Medical  School,  have  proven  satisfactory  and  have  been 
incorporated  in  this  section  which  is  to  be  found  in  the  Appendix. 

Every  effort  has  been  made  to  keep  the  book  within  the  limits  of  a 
pocket  manual. 

Owing  to  my  absence  from  the  United  States  I  have  to  thank  Dr. 
Charles  S-.  Butler  for  correcting  the  proof.  For  the  revision  of  the 
index  I  am  indebted  to  Mr.  John  P.  Griest. 

E.  R.  S. 


PREFACE  TO  THE  FIRST  EDITION. 


WHILE  a  member  of  the  Naval  Examining  Board  and  examiner  in 
bacteriology  and  clinical  microscopy,  I  have  during  the  past  six  years 
had  an  opportunity  to  judge  of  the  qualifications  of  several  hundred 
graduates  of  the  various  medical  schools  of  the  country  from  the  stand- 
point of  practical  application  in  the  laboratory  of  that  which  they  had 
learned  as  undergraduates. 

More  particularly  I  have  made  it  a  point  to  ascertain  from  the  suc- 
cessful candidates,  while  under  instruction  at  the  Naval  Medical  School, 
the  features  of  their  laboratory  courses,  which  had  seemed  to  them 
most  practical;  such  methods  being  subsequently  tested  in  our  own 
class  work. 

As  a  result  I  have  endeavored  to  incorporate  in  this  manual  methods 
which  have  been  submitted  to  the  criticism  of  postgraduate  students 
from  all  the  leading  medical  schools  of  the  country,  and  which  have 
been  considered  by  them  adapted  to  the  requirements  of  practical, 
speedy,  and  satisfactory  clinical  laboratory  diagnosis. 

For  the  laboratory  worker  the  most  valuable  asset  is  common  sense 
and  he  mifst  be  able  to  bring  to  mind  the  possibilities  of  the  production 
of  various  artefacts  and  results  from  trivial  errors  in  technic.  It  has 
been  my  object  to  point  out  where  such  mistakes  may  arise,  the  reasons 
for  obtaining  results  differing  from  those  ordinarily  obtained  and  the 
means  employed  to  eliminate  as  far  as  possible  such  results. 

We  are  too  apt  to  neglect  the  trivial  details  of  stains,  reaction  of 
media,  and  the  like,  yet  it  is  only  when  every  detail  of  technic  has  been 
rigidly  carried  out  that  we  are  in  a  position  to  judge  of  the  significance 
of  an  object  observed  in  a  microscopical  preparation. 

In  bacteriology,  candidates  were  frequently  able  to  give  the  cul- 
tural and  morphological  characteristics  of  all  the  important  pathogenic 
organisms,  yet  when  it  was  required  of  them  to  outline  the  procedure 
by  which  they  would  differentiate  members  of  the  typhoid-colon  groups 
when  encountered  in  a  plate  made  from  feces,  the  problem  appeared 
to  them  impossible.  They  possessed  the  information,  but  did  not 
know  how  to  apply  it. 

ix 


X  PREFACE    TO    THE    FIRST   EDITION 

In  practical  work,  organisms  can  only  be  separated  culturally  by  the 
use  of  Keys  and  for  this  reason  Keys  are  given  at  the  beginning  of  each 
division  of  bacteria.  These  enable  one  to  quickly  place  the  organism 
isolated  in  its  respective  group.  Only  methods  of  differentiation  which 
are  applicable  in  a  physician's  private  laboratory  are  given.  Practical 
methods  for  making  the  final  identification  by  agglutination  or  other 
immunity  tests  are  described.  Technic  for  immunizing  animals  to 
furnish  such  sera  is  given  in  detail. 

The  giving  of  the  cultural  characteristics  in  a  systematic  tabulated 
Key  gives  space  in  the  notes  for  presenting  the  salient  points  in  the 
pathological  and  epidemiological  aspects  of  each  organism. 

I  have  endeavored  to  give  a  scientific  yet  practical  classification  of 
the  important  pathogenic  moulds,  a  subject  about  which  there  exists 
greater  confusion  in  the  minds  of  students  than  for  any  other.  In  the 
nomenclature  I  have  followed  Gedoelst's  "Les  Champignons  Parasites." 

In  the  chapter  on  Media  Making,  it  is  believed  that  anyone  after 
reading  this  section  and  following  the  instructions  will  be  able  to  satis- 
factorily and  without  the  adjuncts  of  a  large  laboratory  make  any  kind 
of  media.  The  directions  as  to  titrations  are  given  in  detail  because 
it  is  beginning  to  be  recognized  that  reaction  of  media  in  bacteriology  is 
of  as  great  importance  as  staining  is  in  blood  work. 

The  section  on  Blood  Work  is  practical  and  gives  a  method  for  mak- 
ing a  Romanowsky  stain  which  is  quick  and  reliable.  The  chapter  on 
Normal  and  Pathological  Blood  gives  in  a  few  pages  the  more  important 
points  to  be  born'e  in  mind  in  considering  a  possible  diagnosis. 

While  there  is  no  difference  between  the  laboratory  requirements  of 
medical  work  in  the  tropics  and  that  in  temperate  climates,  unless  by 
reason  of  such  measures  of  diagnosis  being  indispensable  in  the  tropics, 
it  has,  however,  been  my  endeavor  to  treat  every  tropical  question, 
whether  in  blood  work,  bacteriology,  or  animal  parasitology,  in  a  more 
complete  way  than  is  usual  in  manuals  of  this  character.  Therefore  it 
is  believed  that  his  little  book  will  be  of  great  service  to  the  laboratory 
worker  in  the  tropics. 

It  is  only  from  working  under  Doctor  Charles  W.  Stiles  in  his  course 
of  laboratory  instruction  in  Animal  Parasitology  in  the  United  States 
Naval  Medical  School  that  I  feel  justified  in  presenting  a  concise  out- 
line of  the  subjects  in  medical  zoology  which  appear  to  me  to  be  most 
important  for  the  physician. 

The  system  of  arranging  tables,  showing  the  families,  genera,  etc., 


PREFACE    TO    THE    FIRST    EDITION  XI 

in  which  each  species  belongs  will,  it  is  believed,  greatly  simplify  the 
matter  of  classification  for  the  medical  student.  The  points  given 
under  each  parasite  are  believed  to  be  practical  ones.  When  a  parasite 
has  only  been  reported  for  man  two  or  three  times,  very  little  space  is 
given  to  it. 

Part  IV  summarizes  the  various  infections  which  may  be  found  in 
different  organs  or  excretions  of  the  body  and  embraces  both  bacterial 
and  animal  parasites.  Practical  methods  for  examining  material  are 
also  given. 

The  chapter  on  Immunity,  in  which  the  theoretical  side  is  immedia- 
ately  illustrated  by  the  practical  application  will  tend  to  simplify  this 
bug-bear  of  the  medical  student. 

The  illustrations  have  been  selected  with  a  view  to  bringing  out 
points  which  are  difficult  to  state  briefly  in  the  text,  and  furthermore 
they  have  been  grouped  together  so  that  comparison  of  similar  parasites 
is  possible  without  turning  from  page  to  page. 

I  have  in  particular  to  thank  Hospital  Steward  Ebeling  of  the  Navy 
for  his  care  in  bringing  out  such  details. 

By  reason  of  the  authority  of  Braun,  it  has  been  considered  sufficient 
to  give  in  the  tables  only  the  proper  zoological  name  of  the  parasite  as 
given  in  the  1908  German  edition.  The  synonyms  have  been  omitted 
for  consideration  of  space. 

The  works  chiefly  consulted  in  addition  to  that  of  Braun  have  been: 
Albutt's  System  of  Medicine;  Osier's  System  of  Medicine;  Muir  and 
Ritchie's  Bacteriology;  Mense's  Tropenkrankheiten ;  Blanchard's  Les 
Moustiques;  Guiart  and  Grimbert's  Diagnostic;  Ehrlich's  Studies  in 
Immunity;  Stephens  and  Christopher's  Practical  Study  of  Malaria; 
Daniel's  Laboratory  Studies  in  Tropical  Medicine;  Manson's  Tropical 
Diseases;  Gedoelst's  Les  Champignons  Parasites;  Neveu-Lemaire 
Parasitologie  Humaine;  Chester's  Determinative  Bacteriology;  Leh- 
mann  and  Neumann's  Bacteriology. 

E.  R.  S. 


CONTENTS. 


PART  I.     BACTERIOLOGY. 

CHAPTER  I.— APPARATUS. 

The  Microscope,  i; — Apparatus  for  sterilization,  5; — Cleaning  glassware, 
8; — Concave  slides,  fermentation  tubes,  10. 

CHAPTER  II.— CULTURE  MEDIA. 

Nutrient  bouillon,  17; — Standardization  of  reaction,  19; — Sugar-free 
bouillon,  20; — Glycerine  bouillon,  21; — Peptone  solution,  21; — Nutrient 
agar,  21; — Glycerine  agar  egg  medium,  23; — Gelatine,  23; — Litmus  milk, 
24; — Potato, — 25; — Blood  serum,  25; — Blood  agar,  26; — Bile  and  faeces 
media,  26; — Culture  media  for  protozoa,  29. 

CHAPTER  III.— STAINING  METHODS. 

Loffler's  methylene  blue,  33; — Carbol  fuchsin,  33; — Gram's  method,  33; — 
Acid-fast  staining,  35;— Neisser's  stain,  36;— Capsule  staining,  37;— Fla- 
gella  staining,  38; — Spore  staining,  39; — Staining  of  protozoa,  39. 

CHAPTER  IV.— STUDY  AND  IDENTIFICATION  OF  BACTERIA.     GENERAL  CONSIDERA- 
TIONS. 
Methods  of  isolating  bacteria,  41; — Classification,  43; — Use  of  keys,  45. 

CHAPTER  V. — STUDY  AND  IDENTIFICATION  OF  BACTERIA.     Cocci. 

Key,  49; — Streptococci,  50;— Sarcinae,  53; — Staphylococci,  54; — Pneumo- 
coccus,  55; — Gram-negative  cocci,  57. 

CHAPTER    VI. — STUDY    AND    IDENTIFICATION    OF    BACTERIA.     SPORE-BEARING 
BACILLI. 

Key,  63; — Anthrax,  64; — Cultivation  of  anaerobes,  67; — Malignant  cedema, 
69; — B.  botulinus,  70; — B.  tetani,  72; — B.  aerogenes  capsulatus,  75. 

CHAPTER  VII. — STUDY  AND  IDENTIFICATION  OF  BACTERIA.     BRANCHING,  CURV- 
ING BACILLI.    MYCOBACTERIA.     CORNYEBACTERIA. 

Acid-fast  bacilli,  76; — Tubercle  bacillus,  78; — Leprosy  bacillus,  82; — 
Non-acid-fast  branching  bacilli,  84; — B.  mallei,  84; — B.  diphtheriae,  85; 
— Hofman's  bacillus,  89; — B.  xerosis,  89. 

CHAPTER  VIII.— STUDY  AND  IDENTIFICATION  OF  BACTERIA. 

Gram-negative  bacilli,  Hemophilic  bacteria,  91; — Influenza  bacillus,  92; — 
Friedlander's  bacillus,  95; — Plague,  95; — Eberth,  Gartner,  and  Escherich 
groups,  99; — Typhoid,  100; — Dysentery,  105; — Chromogenic  bacilli,  109. 

xiii 


XIV  CONTENTS 

CHAPTER  IX. — STUDY  AND  IDENTIFICATION  or  BACTERIA. 
Spirilla,  112; — Chlolera,  112. 

CHAPTER  X. — STUDY  AND  IDENTIFICATION  OF  MOULDS,  117. 

CHAPTER  XI. — BACTERIOLOGY  OF  WATER,  AIR,  AND  MILK. 
Water,  127; — Milk,  132; — Air,  135. 

CHAPTER  XII.— PRACTICAL  METHODS  IN  IMMUNITY. 

Methods  of  obtaining  immune  sera,  141; — Agglutination  tests,  143; — 
Deviation  of  the  Complement,  145; — Fixation  of  the  Complement,  146; — 
The  Wassermann  reaction,  147; — Opsonic  power  and  preparation  of  vac- 
cines, 156; — Anaphylaxis,  161. 

PART  II.  STUDY  OF  THE  BLOOD. 

CHAPTER   XIII.— MlCROMETRY  AND  BLOOD  PREPARATIONS. 

Micrometry,  169; — Haemoglobin  estimation,  172; — Counting  blood,  174; — 
Study  of  fresh  blood,  177; — Blood  films,  179; — Staining  blood  films,  179; — 
lodophilia,  185; — Occult  blood,  186. 

CHAPTER  XIV.— NORMAL  AND  PATHOLOGICAL  BLOOD. 

Color  index,  188; — Red  cells,  188; — White  cells,  190; — Eosinophilia,  196; — 
Leukocytosis,  197; — Lymphocytosis,  199; — Diseases  with  a  normal  leuko- 
cyte count,  199; — The  primary  anaemias,  199; — Secondary  anaemias,  201; 
—The  leukemias,  202. 

PART  III.     ANIMAL  PARASITOLOGY. 
CHAPTER  XV. — CLASSIFICATION  AND  METHODS,  211. 

CHAPTER  XVI.— THE  PROTOZOA. 

Rhizopoda,  218; — Flagellata,  222; — Infusoria,  231; — Sporozoa,  233; — 
The  malarial  parasite,  235. 

CHAPTER  XVII.— THE  FLAT  WORMS. 

Flukes,  245; — Liver  flukes,  247; —  Intestinal  flukes,  249; — Lung  flukes, 
250; — Blood  flukes,  251; — Cestodes,  253; — Somatic  taeniasis,  259. 

CHAPTER  XVIIL— THE  ROUND  WORMS. 

Filariidae,  265; — Key  to  filarial  larvae,  269; — Trichinosis,  271; — Hook- 
worms, 274; — Ascaridae,  277; — Leeches,  280. 

CHAPTER  XIX.— THE  ARACHNOIDEA. 

The  mites,  282;— The  ticks,  285;— The  Linguatulidae,  289. 

CHAPTER  XX.— THE  INSECTS. 

The  Pediculidae,  292; — The  Diptera,  298; — Biting  flies,  299. 

CHAPTER  XXL— THE  MOSQUITOES. 

Dissection  of  mosquitoes,  311; — Differentiation  of  Culicinae  and  Anophe- 
linae,  312; — Classification  of  Culicidae,  313. 

CHAPTER  XXII.— THE  POISONOUS  SNAKES,  317. 


CONTENTS  XV 

PART  IV.     CLINICAL  BACTERIOLOGY    AND  ANIMAL 

PARASITOLOGY  OF  THE  VARIOUS  BODY 

FLUIDS  AND  ORGANS. 

CHAPTER  XXIII. — DIAGNOSIS  or  INFECTIONS  OF  THE  OCULAR  REGION,  325. 

CHAPTER  XXIV. — DIAGNOSIS  OF  INFECTIONS  OF  THE  NASAL  CAVITIES,  328. 

CHAPTER  XXV. — EXAMINATION  OF  BUCCAL  AND  PHARYNGEAL  MATERIAL,  330. 

CHAPTER  XXVI.— EXAMINATION  OF  SPUTUM,  333. 

CHAPTER  XXVII.— THE  URINE,  337. 

CHAPTER  XXVIII.— THE  FAECES,  345. 

CHAPTER  XXIX.— BLOOD  CULTURES  AND  BLOOD  PARASITES,  351. 

CHAPTER  XXX.— THE  STOMACH  CONTENTS,  354. 

CHAPTER  XXXI.— EXAMINATION  OF  Pus,  355. 

CHAPTER  XXXII.— SKIN  INFECTIONS,  357. 

CHAPTER  XXXIII.— CYTODIAGNOSIS,  359. 

CHAPTER  XXXIV.— RABIES  AND  VACCINIA,  362. 

APPENDIX. 

PREPARATION  OF  TISSUES  FOR  EXAMINATION  IN  MICROSCOPIC  SECTIONS,  371. 

MOUNTING  AND  PRESERVATION  OF  ANIMAL  PARASITES,  377. 

PREPARATION  OF  NORMAL  SOLUTIONS,  380. 

DISEASES  OF  UNKNOWN  ETIOLOGY,  381. 

CHEMICAL  EXAMINATION  OF  THE  URINE,  383. 

CHEMICAL  EXAMINATION  OF  THE  GASTRIC  CONTENTS,  392. 

CHEMICAL  TESTS  OF  F^CES,  393. 

DISINFECTANTS  AND  INSECTICIDES,  394. 


BACTERIOLOGY,    BLOOD-WORK  AND 
ANIMAL   PARASITOLOGY. 


CHAPTER  I. 
APPARATUS. 

THE  MICROSCOPE. 

THE  most  important  piece  of  apparatus  for  the  laboratory  worker 
is  the  microscope.  Very  satisfactory  microscopes  can  be  purchased  in 
this  country.  Instruments  of  standard  German  make  are  in  use  in 
many  laboratories  and  appear  to  give  general  satisfaction.  It  is  impos- 
sible to  do  good  microscopical  work  unless  the  microscope  gives  and 
continues  to  give  good  definition  and  the  working  parts  remain  firm. 
Folding  microscope  stands  are  now  made  which  are  perfectly  satisfac- 
tory, such  instruments,  however,  have  only  the  advantage  of  occupying, 
less  space  in  a  case  so  that  unless  the  question  of  compactness  is  involved, 
as  in  an  outfit  for  the  military  services  or  for  a  microscopist  who  travels 

about  a  great  deal,  the  ordinary  rigid  horseshoe  base  is  to  be  preferred. 

• 
A  mechanical  stage  is  almost  a  necessity  in  connection  with  blood- work  and  its 

use  is  advantageous  in  bacterial  preparations.  For  the  study  of  tissue  sections  the 
moving  of  the  slide  with  the  ringers  is  preferable.  Therefore,  the  mechanical 
stage  should  be  capable  of  ready  attachment  or  removal.  For  the  examination  of 
colonies  growing  in  Petri  dishes  we  also  use  the  stage  unencumbered  with  the  me- 
chanical stage.  A  triple  or  quadruple  nose-piece,  according  to  the  number  of 
objectives  used,  is  also  indispensable. 

Objectives. — To  meet  the  demands  of  clinical  microscopy  there 
should  be  three  objectives,  preferably  a  i6-mm.  (2/3 -in.),  a  4-mm. 
(i/6-in.)  and  a  2-mm.  (i/i2-in.)  homogeneous  oil  immersion.  The 
Zeiss  AA  is  a  ly-mm.  objective,  and  the  Leitz  No.  3,  an  i8-mm.  one. 
The  Zeiss  D  is  about  4. 2-mm.  and  the  Leitz  No.  6,  a  4. 4-mm.  A  dust- 
proof  quadruple  nose-piece  with  four  objectives  will  be  found  a  great 
convenience  (in  addition  to  the  2/3 -in.  and  i/i2-in.  objectives,  a  i/4-in. 


2  APPARATUS 

for  urine  and  blood  counting,  with  a  i/8-in.  for  examining  hanging-drop 
preparations  and  for  quick  examination  of  blood  smears).  An  apo- 
chromatic  objective  costs  about  three  times  as  much  as  an  achromatic 
one  and,  except  in  photographic  work,  has  little  if  any  advantage. 

As  regards  oculars  (eye-pieces)  a  No.  2  and  a  No.  4  will  best  meet 
the  requirements.  For  high  magnification  a  No.  8  may  be  of  service. 
The  Zeiss  oculars  are  numbered  according  to  the  amount  they  increase 
the  magnification  given  by  the  objective;  thus  a  No.  2  increases  the 
magnification,  given  by  the  objective  alone,  twice;  a  No.  8,  eight  times. 
Some  oculars  are  classified  according  to  the  equivalent  focal  distance, 
and  are  referred  to  as  i/2-in.,  i-in.,  and  2-in.  oculars. 

The  oculars  in  common  use  are  known  as  negative  oculars,  by  which  is  meant 
an  ocular  in  which  the  lower  lens  (collective)  assists  in  forming  the  real  inverted  image 
which  is  focused  at  the  level  of  the  diaphragm  within  the  ocular.  When  using  a  disc 
micrometer,  it  is  supported  by  this  diaphragm,  and  the  outlines  of  the  image  are 
cut  by  the  rulings  on  the  glass  disc,  and  so  we  are  enabled  to  measure  the  size  of  the 
object  examined.  The  measurement  of  various  bacteria,  blood-cells,  and  parasites  is 
exceedingly  simple  and  assists  greatly  in  the  study  of  bacteria,  and  is  indispensable 
in  work  in  animal  parasitology.  (For  details  of  micrometry  see  section  on  blood- 
work.)  When  an  ocular  is  termed  positive,  it  refers  to  an  ocular  which  acts  as  a 
simple  microscope  in  magnifying  the  image,  the  image  being  formed  entirely  by  the 
objective  and  being  located  below  the  ocular. 

Objectives  are  usually  designated  by  their  equivalent  focal  distance.  It  is  im- 
portant to  remember  that  the  equivalent  focal  distance  does  not  represent  the  work- 
ing distance  of  an  objective,  by  which  is  meant  the  distance  from  the  upper  surface 
of  the  cover-glass  to  the  lower  surface  of  the  objective.  Thus  a  i/4-in.  objective 
may  have  to  be  approached  to  the  object  so  that  the  distance  intervening  may  be 
only  1/6  in.  or  even  less.  This  explains  the  frequent  inability  to  locus  an  object 
when  a  high-power  dry  objective  (i/6-in.  or  i/8-in.)  is  used  with  a  rather  thick  cover- 
glass — the  objective  possibly  having  a  short  working  distance,  so  that  the  thickness 
of  the  cover-glass  does  not  allow  of  any  free  working  distance. 

Instrument  makers  generally  specify  the  thickness  of  cover-glass  to  be  used  with 
a  certain  tube  length,  but  as  a  practical  matter  it  will  be  found  convenient  to  use 
No.  i  (very  thin)  cover-glasses.  The  principal  objection  to  these  is  that  they  are 
more  fragile  than  the  No.  2,  but  with  a  little  practice  in  cleaning  cover-glasses  this 
is  negligible.  Immersion  lenses  are  less  affected  than  dry  lenses  by  the  question  of 
a  certain  thickness  of  cover-glasses  for  a  certain  tube  length. 

One  of  the  most  fruitful  causes  of  the  crushing  of  microscopical 
objects  and  the  overlying  cover-glass  or,  what  is  far  more  important, 
the  breaking  of  the  cover-glass  of  a  hanging-drop  preparation  and 
consequent  risk  of  infection  is  the  attempt  to  focus  with  the  fine  adjust- 
ment. It  should  be  an  invariable  rule  for  the  worker  to  bring  his  object- 


USE   OF   THE   MICROSCOPE  3 

ive  practically  into  contact  with  the  upper  surface  of  the  cover-glass, 
then  using  the  coarse  adjustment  (rack  and  pinion)  to  slowly  elevate 
the  objective,  looking  through  the  eye-piece  at  the  same  time.  In 
other  words,  obtain  focus  with  the  coarse  adjustment  and  maintain  it 
with  the  fine  adjustment  (micrometer  screw).  The  fine  adjustment 
should  only  be  used  after  the  focus  is  obtained. 

In  using  the  oil-immersion  objective  always  dip  the  lens  in  the  oil  and  practically 
touch  the  cover-glass — the  eye  being  at  a  level  with  the  stage — before  beginning 
to  focus.  With  the  coarse  adjustment  one  can  feel  the  contact  with  the  cover- 
glass,  which  is  impossible  with  the  fine  adjustment.  It  saves  time  and  disappoint- 
ment to  make  a  preliminary  examination  of  a  preparation  requiring  the  high  dry 
or  immersion  lens  with  a  low  power  (2/3-in.)  before  employing  the  higher  power; 
in  this  way  we  locate  or  center  a  suitable  field  for  study. 

It  will  be  observed  that  objectives  frequently  have  their  numerical  aperture 
marked  on  them.  This  is  expressed  by  the  letters  N.A.  From  a  practical  stand- 
point this  gives  the  relative  proportion  of  the  rays  which  proceeding  from  an  object 
can  enter  the  lens  of  the  objective  and  form  the  image.  Of  course,  the  greater  the 
number  of  rays,  the  greater  the  N.A. ,  the  better  the  definition,  and  consequently 
the  better  the  objective.  Immersion  oil,  having  the  same  index  of  refraction  (1.52) 
as  glass,  would  not  deflect  rays  coming  from  the  object  and  so  prevent  their  enter- 
ing the  objective,  as  would  be  the  case  if  we  used  a  dry  objective  with  an  interven- 
ing air  space.  In  this  case  a  portion  of  the  rays  would  be  turned  aside  by  the  dif- 
ference in  the  refractive  index  of  air.  As  a  rule,  the  higher  the  numerical  aperture, 
the  better  the  objective  and  the  less  the  working  distance.  In  blood  counting,  the 
cover-glass  being  comparatively  thick,  it  may  happen  that  with  a  i/6-in.  of  high 
numerical  aperture  there  may  not  be  sufficient  working  distance  to  bring  the  blood- 
cells  into  focus,  which  could  be  done  with  an  objective  of  lower  numerical  aperture. 
Consequently,  we  must  always  consider  the  matter  of  working  distance  as  well  as 
that  of  numerical  aperture.  The  skill  of  the  optician,  however,  can  obviate  this 
defect  in  an  objective  of  high  numerical  aperture  so  that  it  may  combine  the  qualities 
of  perfect  definition  with  sufficient  working  distance. 

Practical  Points  in  the  Use  of  the  Microscope. — An  important 
matter  in  the  use  of  the  microscope  is  to  get  all  the  details  possible  with 
a  low  power  before  using  a  higher  power.  This,  of  course,  does  not 
apply  to  a  bacterial  preparation  where  it  is  necessary  to  use  a  i/i2-in. 
or  a  high-power  dry  lens.  It  is  well,  however,  in  a  bacterial  or  blood 
preparation  to  first  examine  the  smear  with  the  2/3-in.  objective  in 
order  to  determine  suitable  areas  for  examination  with  the  oil-immer- 
sion objective.  With  tissue  sections  it  is  not  only  advisable  to  begin  the 
study  with  the  lowest  power,  but  even  an  examination  with  the  unaided 
eye  or  with  a  magnifying  glass,  before  using  the  microscope,  will  give  a 
surprising  amount  of  information. 


4  APPARATUS 

After  using  the  oil-immersion  objective  the  lens  should  be  wiped  clean  of  oil 
with  a  strip  of  Japanese  lens  paper  or  with  a  silk  handkerchief.  If  the  oil  should 
dry  on  the  surface  of  the  lens  it  may  be  removed  with  a  drop^of  xylol  on  a  piece  of 
lens  paper.  Immediately  afterward  the  lens  should  be  dried.  Dried  oil  on  a  lens 
often  causes  the  lens  to  be  considered  defective.  Accidental  contact  of  the  dry 
objectives  with  oil  is  not  uncommon  and  should  always  be  thought  of  when  satis- 
factory optical  effects  are  not  obtainable. 

It  is  advisable  to  cultivate  the  use  of  both  eyes  in  doing  microscopical  work. 
When  using  one  eye  the  other  should  be  kept  open  with  accommodation  relaxed. 
It  is  this  squinting  of  the  unemployed  eye  which  so  often  fatigues.  A  strip  of  card- 
board 4  or  5  inches  long,  with  an  opening  to  fit  over  the  tube  of  the  microscope, 
leaving  the  other  end  to  block  the  vision  of  the  unused  eye,  will  prevent  the  strain. 
This  apparatus  can  be  purchased  in  vulcanite. 

A  warm  stage  for  the  study  of  living  protozoa  may  be  extemporized  by  taking 
a  piece  of  copper  about  the  size  of  the  stage  and  with  a  strip  projecting  out  ante- 
riorly for  5  or  6  inches.  The  under  surface  of  the  plate  is  covered  with  flannel  and 
a  hole  about  i  inch  in  diameter  cut  out  of  the  center.  The  proper  amount  of  heat 
is  applied  by  a  flame  impinging  on  the  tongue-like  projection  of  the  copper  plate. 

Direct  sunlight  or  excessively  bright  light  is  to  be  avoided.  If  such  conditions 
must  exist  a  white  shade  or  muslin  curtain  drawn  across  the  window  is  a  necessity. 
Light  from  the  north  and  from  a  white  cloud  is  the  most  desirable.  South  of  the 
equator  a  southern  light.  In  the  tropics  a  piece  of  plate  glass  fitted  into  the  lower 
part  of  a  wire  screen  frame  gives  good  lighting,  keeps  out  dust,  and  does  not  interfere 
greatly  with  the  circulation  of  the  air. 

The  technic  in  connection  with  proper  illumination  is  probably  more  important 
than  any  other  point;  unless  the  light  is  utilized  to  the  best  advantage,  the  best  re- 
sults cannot  be  obtained.  In  examining  fresh  blood  preparations  or  hanging  drops 
the  concave  mirror  should  be  used  and  the  light  almost  shut  off  by  the  iris  dia- 
phragm so  as  to  give  a  contour  picture.  In  examining  a  stained  blood  or  bacterial 
preparation,  the  Abbe  condenser  should  be  properly  focused  so  as  to  best  illuminate 
the  stained  film.  In  many  instruments  set-screws  are  provided  which  check  the 
elevation  of  the  Abbe  condenser  when  the  proper  focus  is  reached.  Inasmuch  as 
the  light  from  the  condenser  should  come  to  a  focus  exactly  level  with  the  object 
studied,  it  is  evident  that  a  fixed  position  for  the  condenser  would  not  answer  when 
slides  of  different  thickness  were  used.  Always  use  the  plane  mirror  when  examin- 
ing stained  bacterial  or  blood  films,  as  a  color  image  is  desired.  Ordinarily  in  ex- 
amining tissue  sections,  the  Abbe  condenser  should  either  be  put  out  of  focus  by 
racking  down  or  by  the  use  of  the  concave  mirror  and  the  narrowing  of  the  aperture 
of  the  iris  diaphragm.  Swing-out  condensers  are  now  made  which  are  very  con- 
venient. The  proper  employment  of  illumination  only  comes  with  experience,  and 
one  should  continue  to  manipulate  his  mirrors,  diaphragm,  and  condenser  until 
the  best  result  is  obtained.  Then  study  the  specimen. 

For  microscopical  work  in  a  laboratory  not  properly  supplied  with  windows  or 
for  night  work  the  frosted  incandescent  bulb  is  very  satisfactory. 

Dark  Ground  Illumination. — Very  valuable  information,  especially 
as  regards  the  detection  of  treponemata  in  material  from  hard  chancres 


DARK   GROUND   ILLUMINATION  5 

or  mucous  patches,  may  be  obtained  by  the  use  of  dark  ground  illumina- 
tion.    There  are  many  different  types  of  apparatus  for  this  purpose. 

The  bacteria  or  spirochaetes  are  intensely  illuminated  and  show  as 
brilliant  silvery  objects  in  contrast  to  the  dark  background. 

When  the  morphological  details  of  a  brightly  illuminated  object  in  the  dark 
field  can  be  distinctly  observed  it  is  proper  to  use  the  term  dark  ground  illumina- 
tion. When  only  particles,  usually  surrounded  by  bright  and  dark  rings,  and  not 
showing  any  structure,  are  observed  in  the  dark  field  the  proper  designation  is  ultra- 
microscopic.  An  apparatus  using  only  the  short  waves  of  the  ultra-violet  spectrum 
enables  one  to  observe  particles  no  larger  than  i/io  of  a  micron.  For  this  appa- 
ratus it  is  necessary  to  employ  photographic  plates.  In  using  the  i/i 2-inch  ob- 
jective with  dark  ground  illumination  a  funnel-like  base  is  supplied  on  which  we 
screw  the  nickle  plated  front  mount  of  the  objective.  Before  using  the  dark- field 
apparatus  it  must  be  centered  with  a  low  power.  This  is  carried  out  by  getting 
concentric  rings  parallel  with  the  circle  of  the  microscopic  field.  Immersion  con- 
tact b.etween  the  front  surface  of  the  Abbe  condenser  and  the  under  surface  of  the 
slide  carrying  the  preparation  must  be  made  before  focussing  the  i/i2th  objective. 
As  a  source  of  illumination  we  may  use  a  small  arc-lamp  or  a  Nernst  lamp  or  an 
incandescent  gas  lamp.  In  using  an  arc-lamp  one  must  have  a  suitable  rheostat 
according  to  the  electrical  current  employed.  Information  as  to  voltage  and  nature 
of  current  must  be  given  the  one  supplying  the  apparatus. 

In  making  preparations  the  slides  and  cover-slips  should  be  scrupulously  clean 
and  the  material  thinly  spread  out  and  free  of  bubbles. 

APPARATUS  FOR  STERILIZATION. 

For  the  purpose  of  sterilizing  glassware,  media,  and  old  cultures 
there  are  three  methods  ordinarily  employed.  The  hot-air  sterilizer, 
in  which  a  temperature  of  about  150°  C.  is  maintained  for  one  hour,  is 
ordinarily  used  for  the  sterilization  of  Petri  dishes,  test-tubes,  pipettes, 
etc.  If  the  temperature  is  allowed  to  go  too  high,  there  is  danger  of 
charring  the  cotton  plugs  and  also  of  causing  the  development  of  an 
empyreumatic  oil  which  makes  the  plugs  unsightly  and  causes  them  to 
stick  to  the  glass.  Again  we  must  be  careful  not  to  open  the  door  until 
the  temperature  has  fallen  to  60°  C.,  otherwise  there  is  danger  of  crack- 
ing the  glassware.  Where  gas  is  not  obtainable,  the  hot-air  sterilizer  is 
not  a  very  satisfactory  apparatus. 

The  Arnold  sterilizer  is  to  be  found  everywhere  and  can  be  used  on 
blue-flame  kerosene-oil  stoves  as  readily  as  with  gas  burners.  The 
most  convenient  form,  but  more  expensive,  is  the  Boston  Board  of 
Health  pattern.  The  ordinary  pattern,  with  a  telescoping  outer  por- 
tion, answers  all  purposes,  however.  In  the  Arnold,  sterilization  is 


6  APPARATUS 

effected  by  streaming  steam  at  100°  C.  It  is  usual  to  maintain  this 
temperature  for  fifteen  to  twenty-five  minutes  each  day  for  three  suc- 
cessive days.  The  success  of  this  procedure — fractional  sterilization — 
is  due  to  the  fact  that  many  spores  which  were  not  killed  at  the  first 
steaming  have  developed  into  vegetative  forms  within  twenty-four 


FIG.  i. — Dressing  sterilizer  showing  cylinder  containing  water  (K)  heated  either 
by  gas  or  Primus  kerosene  lamps. 

hours,  and  when  the  steam  is  then  applied  such  forms  are  destroyed. 
Experience  has  shown  that  all  the  spores  have  developed  by  the  time 
of  the  third  steaming,  so  that  with  this  final  application  of  heat  we 
secure  perfect  sterilization. 

It  is  customary  to  use  the  Arnold  for  sterilizing  gelatin  and  milk 


STERILIZATION  7 

media,  even  when  the  autoclave  is  at  hand,  the  idea  being  that  the 
greater  heat  of  the  autoclave  may  interfere  with  the  quality  of  such 
media.  The  most  convenient  autoclave  is  the  horizontal  type,  such  as 
is  to  be  found  everywhere  for  the  sterilization  of  surgical  dressings. 

The  source  of  heat  may  be  either  gas,  the  Primus  kerosene-oil  lamp  or  steam 
from  an  adjacent  boiler.  More  recently  a  method  of  employing  kerosene,  gaso- 
lene, or  alcohol  with  a  gravity  system  has  been  perfected.  During  the  past  6  years, 
in  the  laboratory  of  the  U.  S.  Naval  Medical  School,  we  have  been  using  a  dressing 
sterilizer,  made  by  the  American  Sterilizer  Co. ,  with  which  it  has  been  possible  to 
most  satisfactorily  carry  out  all  kinds  of  sterilization,  thus  doing  away  with  the 
use  of  the  Arnold  and  the  hot-air  sterilizer.  It  is  impossible  to  sterilize  ordinary 
fermentation  tubes  in  the  autoclave  on  account  of  the  boiling  up  of  the  media  and 
wetting  of  the  plugs.  This  is  still  done  with  the  Arnold.  By  use  of  the  Durham 
tubes — which  are  to  be  preferred,  except  for  gas  analysis — sugar  media  can  be  thus 
sterilized. 

Should  a  small  bubble  remain  in  the  top  of  the  small  inverted  inner  tube  after 
removal  from  the  autoclave,  one  may  make  a  mark  with  a  grease  pencil  at  the  line 
of  the  bubble;  or,  if  preferred,  the  basket  of  Durham  tubes  can  be  heated  to  boiling 
for  ten  minutes  in  a  pan  of  water  or  in  the  Arnold  when,  after  cooling,  the  bubSle 
will  be  found  to  have  disappeared. 

Glassware  will  come  out  from  such  an  autoclave  with  wrappers  as  dry  and  plugs 
of  the  test-tubes  as  stopper-like  as  could  be  effected  in  a  hot-air  sterilizer. 

The  objection  which  exists  in  the  use  of  some  autoclaves,  as  regards  condensa- 
tion on  dressings  or  apparatus,  does  not  exist  in  this  type.  The  mechanism,  by 
which  the  inner  and  outer  chambers  are  connected  and  disconnected,  and  that  for 
vacuum  production,  rests  in  the  simple  turning  of  a  lever  from  mark  to  mark.  We 
have  been  able  with  a  gas  burner  to  obtain  a  pressure  of  15  pounds  in  less  than  ten 
minutes.  In  sterilizing  test-tubes  we  place  them  in  small  rectangular  wire  baskets, 
6X5X4  in.  These  baskets  are  to  be  preferred  to  round  ones,  as  they  pack  more 
satisfactorily  in  the  refrigerator  used  for  storing  media.  In  sterilizing  flasks,  test- 
tubes,  Petri  dishes,  throat  swabs,  pipettes,  etc.,  it  has  been  our  custom,  after  ex- 
posing to  20  pounds'  pressure  for  twenty  minutes,  to  produce  a  vacuum  for  two  or 
three  minutes;  then  with  the  steam  in  the  outer  jacket  for  a  few  minutes  to  thor- 
oughly dry  the  articles  in  the  disinfecting  chamber.  The  valve  to  the  inner  chamber 
is  then  opened  to  break  the  vacuum;  the  door  is  now  opened,  and  the  articles  re- 
moved in  as  dry  a  state  as  if  they  had  been  in  the  hot-air  sterilizer.  Articles,  how- 
ever, can  be  thoroughly  dried  without  the  use  of  a  vacuum,  simply  allowing  the  steam 
to  remain  in  the  outer  jacket  with  the  steam  cut  off  from  the  inner  chamber. 

PRESSURE  AND  TEMPERATURE  TABLE. 

5  pounds' pressure,  107. 7°  C.,  226°  F. 

10  pounds'  pressure,  n5-5°  C. ,  240°  F. 

15  pounds'  pressure,  121.6°  C. ,  250°  F. 

20  pounds' pressure,  126.6°  C.,  260°  F. 

25  pounds'  pressure,  JSQ-S0  C. ,  267°  F. 

30  pounds'  pressure,  134-4°  C.,  274°  F. 


8  APPARATUS 

All  such  articles  as  Petri  dishes,  pipettes,  swabs,  etc.,  are  wrapped  in  cheap 
quality  filter-paper,  making  a  fold  and  turning  in  the  ends  as  is  done  in  a  druggist's 
package.  Old  newspapers  answer  well  for  this  purpose.  The  sterile  swab  can  be 
used  for  many  purposes  in  the  laboratory.  They  are  most  easily  made  by  taking 
a  piece  of  copper  wire  about  8  inches  long,  flattening  one  end  with  a  stroke  of  a 
hammer,  then  twisting  a  small  pledget  of  plain  absorbent  cotton  around  the  flat- 
tened end.  After  wrapping,  the  swabs  are  sterilized  in  bunches.  We  not  only 
use  them  for  getting  throat  cultures,  but  in  addition  for  culturing  faeces,  pus,  or 
other  such  material.  The  pus  is  obtained  with  a  swab,  which  material  is  then  dis- 
tributed in  a  tube  of  sterile  bouillon  or  water.  With  the  same  swab  the  surface  of 
an  agar  plate  is  successively  stroked.  This  method  is  almost  as  satisfactory  as  the 
German  one  of  using  bent  glass  rods  for  this  purpose.  Everyone  has  encountered 
the  difficulties  attendant  upon  the  bending  of  platinum  wires  and  also  the  possi- 
bility of  destroying  your  organisms  by  an  insufficiently  cooled  wire. 


CLEANING  GLASSWARE. 

It  is  a  routine  in  our  laboratory  for  everything  to  go  through  the 
sterilizer  at  125°  C.  before  anything  else  is  done.  This  is  a  safe  rule 
when  dealing  with  dangerous  pathogenic  organisms  (especially  tetanus 
and  anthrax). 

As  soon  as  taken  out  of  the  sterilizer  the  contents  are  emptied,  and  the  tube  or 
dishes  placed  in  a  i  %  solution  of  washing  soda  and  boiled.  This  thoroughly  cleans 
them.  As  the  washing  soda  slightly  raises  the  boiling-point  and  also  makes  the 
spores  more  penetrable,  it  would  appear  that  under  ordinary  circumstances,  it 
would  be  sufficient  to  place  all  contaminated  articles  in  a  dishpan  with  the  soda 
solution,  and  boil  for  at  least  one  hour,  not  using  a  preliminary  sterilization  in  the 
autoclave.  The  tubes  are  now  cleaned  with  a  test-tube  brush,  thoroughly  rinsed 
with  tap  water  and  placed  in  a  i%  solution  of  hydrochloric  acid  for  a  few  minutes; 
then  rinsed  thoroughly  in  water  and  placed  in  test-tube  baskets,  mouth  downward, 
and  allowed  to  drain  over  night.  Some  laboratory  workers  boil  their  test-tubes 
and  other  glassware  in  water  containing  soap  or  soap  powder  and,  after  a  thorough 
rinsing  in  tap  water,  drain.  Hydrochloric  acid  should  not  be  used  after  the  soap 
as  it  will  cause  the  formation  of  an  unsightly  coating  difficult  to  remove.  When 
thoroughly  dry  they  may  be  plugged  and  sterilized.  To  plug  a  test-tube,  pick  out 
a  little  pledget  of  plain  absorbent  cotton  about  2  inches  in  diameter  from  a  roll. 
Place  it  over  the  center  of  the  tube  and  with  a  glass  rod  push  the  cotton  down  the 
tube  about  an  inch.  The  cleaning  fluid  commonly  used  in  laboratories  consists  of 
one  part  each  of  potassium  bichromate  and  commercial  sulphuric  acid  with  ten 
parts  of  water.  This  is  an  excellent  mixture  for  cleaning  old  slides,  etc. ,  especially 
when  grease  or  balsam  is  to  be  gotten  rid  of.  It  is  very  corrosive,  however.  An 
efficient  and  less  corrosive  method  for  cleansing  slides  and  cover-glasses  is  to  leave 
them  over  night  in  an  acetic  acid  alcohol  mixture  (two  parts  of  glacial  acetic  acid 
to  one  hundred  parts  of  alcohol).  After  drying  and  polishing  out  of  this  mixture, 


HANGING  DROP  9 

it  is  well  to  pass  the  slides  and  cover-glasses  through  the  flame  of  a  Bunsen  burner 
or  alcohol  lamp  to  remove  every  vestige  of  grease.  Ordinarily,  rubbing  between 
the  thumb  and  forefinger  with  soap  and  water,  then  drying  with  an  old  piece  of 
linen,  and  finally  flaming  will  yield  a  perfect  surface  for  making  a  bacterial 
preparation. 


FIG.  2. — i,  Inoculation  of  tubes;  2,  plugging  of  tubes;  3,  filling  tubes;  4,  Smith's 
fermentation  tube;  5,  Durham's  fermentation  tube. 

CONCAVE  SLIDES,  FERMENTATION  TUBES. 

The  concave  slide  is  ordinarily  used  for  making  hanging-drop  prep- 
arations for  the  examination  of  bacteria  as  to  motility,  capsules,  size 
and  arrangement.  To  prepare  a  hanging-drop  preparation  for  the 
study  of  motility  it  is  best  to  place  a  loopful  of  the  young  bouillon 


FIG.  3. — Hanging  drop,  over  hollow  ground  slide.     (Williams.) 

culture  or  a  loopful  of  salt  solution  into  which  is  then  emulsified  a 
small  amount  of  growth  from  an  agar  slant,  in  the  center  of  the  cover- 
glass;  now  having  applied  with  a  brush  a  ring  of  vaseline  around  the 


10  APPARATUS 

concave  depression  in  the  slide  we  apply  the  slide  as  a  cover  to  the 
cover-glass  which  latter  adheres  to  the  ring  of  vaseline.  The  completed 
hanging-drop  preparation  can  now  be  turned  over  and  placed  on  the 
stage  of  the  microscope. 

A  substitute  which  is  equally  good  may  be  made  by  spreading  a  ring  or  square 
of  vaseline — smaller  than  the  cover-glass  to  be  used — in  the  middle  of  a  plain  slide. 
Then  putting  a  loopful  of  salt  solution  in  the  center  of  the  space,  and  inoculating 
with  the  culture  to  be  studied,  we  finally  cover  it  with  a  cover-glass,  gently  pressing 
the  margins  down  on  the  vaseline.  This  gives  a  preparation  for  the  study  of  motility 
or  agglutination  which  does  not  dry  out  for  hours,  and  is  easier  to  focus  upon  than 
the  concave  slide  hanging-drop  preparation 

In  examining  a  hanging  drop  first  use  a  low-power  objective  and,  having  brought 


FIG.  4. — Blood  serum  coagulating  apparatus. 

into  focus  the  margin  of  the  drop  as  a  center  line,  change  to  a  r/6-  or  i/8-in.  objective. 
By  this  procedure  a  thin  layer  of  fluid  is  brought  under  the  high  dry  objective  in- 
stead of  the  deeper  layer  in  the  center  of  the  drop.  It  is  not  advisable  to  use  an 
immersion  objective  with  a  hanging-drop  preparation. 

The  light  should  be  cut  down  to  a  minimum  with  the  iris  diaphragm  and  the 
concave  mirror  used.  When  we  have  finished  examining  the  preparation  the  cover- 
glass  should  be  pushed  over  with  the  forceps  so  that  a  corner  projects  and  we  then 
seize  this  with  the  forceps,  lift  up  the  cover-glass  and  drop  it  into  the  disinfecting 
solution  along  with  the  slide. 

The  fermentation  tube  with  a  bulb  and  closed  arm  is  expensive, 
difficult  to  clean,  and  is  easily  broken.  It  is,  however,  convenient  in 
the  determination  of  the  gas  formula  of  an  organism.  It's  use  is 
described  under  water  analysis.  As  a  substitute  in  the  study  of  gas 
production  and  in  water  bacteriology,  the  Durham  tube  is  to  be 
recommended. 

Into  a  test-tube,  about  1X7  in.,  we  introduce  the  special  sugar  media,  then 
drop  down  a  small  test-tube  (1/2X3  mO  with  its  open  end  downward.  Insert  the 


ACID   PROOFING   FOR   DESKS 


II 


plug  of  the  large  tube  and  sterilize.  During  sterilization  the  fluid  enters  the  mouth 
of  the  smaller  tube  and  fills  it,  and  when  the  medium  is  subsequently  inoculated,  if 
gas  forms,  it  appears  in  the  upper  part  of  the  closed  end  of  the  smaller  tube. 

For  inspissating  blood-serum  slants  a  regular  inspissator  is  desirable. 
This  is  nothing  more  than  a  double-walled  vessel,  the  space  between 
the  walls  being  filled  with  water. 

As  a  substitute  one  may  take  the  common  rice  cooker  (double  boiler).  Fill  the 
outer  part  with  water;  and  in  the  inner  compartment  pack  the  serum  tubes  properly 
slanted  on  a  piece  of  wood  or  a  wedge-shaped  layer  of  cotton.  Place  a  weight  on 
the  cover  of  the  inner  compartment  to  sink  it 
into  the  surrounding  water,  and  allow  to  boil 
for  one  or  two  hours.  This  same  apparatus 
may  be  used  for  their  sterilization  on  two  sub- 
sequent days,  but  it  is  better  to  sterilize  in  the 
autoclave  or  Arnold.  As  regards  a  working 
desk,  it  will  be  found  convenient  to  have  an 
arrangement  similar  to  the  ordinary  flat-top 
desk,  with  a  tier  of  drawers  on  each  side.  A 
block  of  wood  with  holes  bored  in  it  to  contain 
dropping-bottles  may  be  placed  in  the  upper 
lef  thand  drawer.  In  this  way  the  stains  are  as 
accessible  as  if  they  encumbered  the  desk.  It  is  advisable  to  paint  the  inside  of  this 
drawer  black  so  that  the  light  may  not  cause  the  staining  reagents  to  deteriorate. 

A  very  popular  method  of  preparing  the  surfaces  of  laboratory 
desks,  sinks,  and  tables  is  the  application  of  the  so-called  "  acid-proofing." 
This  gives  an  ebony-like  finish- which  is  not  affected  by  strong  acids. 

In  using  it  the  surface  of  the  wood  must  be  new  (free  of  any  varnish,  oil,  or 
paint;  if  previously  so  coated  the  surface  must  be  planed). 


FIG.  5. — Rice  cooker. 


Solution  i. 


Potassium  chlorate, 
Cupric  sulphate, 
Water, 


125.0  gms. 
125.0  gms. 
i ooo.o  c.c. 


Apply  two  coats  of  this  solution  at  least  1 2  hours  between  applications.     When 
thoroughly  dry  apply  two  coats  of  solution  No.  2. 


Solution  2. 


Aniline  oil, 
Hydrochloric  acid, 
Water, 


I2O.O  C.C. 

180.0  c.c. 

IOOO.O  C.C. 


When  the  treated  surface  is  thoroughly  dry  apply  one  coat  of  raw  linseed  oil 
with  a  cloth.     After  this  is  dry  wash  with  very  hot  soapsuds. 


1 2  APPARATUS 

An  aspirating  bottle  on  a  shelf  elevated  two  feet,  with  rubber  tubing  and  glass 
tip  leading  to  a  small  aquarium  jar  or  other  desk  receptacle,  makes  a  good  substitute 
for  a  small  sink  and  faucet.  A  Hoffman  screw  clamp  on  the  rubber  tube  controls 
the  flow  of  water. 

Ordinary  glass  salt  cellars  will  be  found  very  useful,  where  the  watch-glass  is 
employed.  They  may  also  be  wrapped,  sterilized,  and  used  to  contain  fluids  for 
inoculating,  etc. 

A  glass-topped  fruit  jar  or  a  specimen  jar  containing  a  disinfecting  solution  for 
contaminated  slides,  etc.,  should  be  on  every  working  desk.  A  good  solution  is  that 
of  Harrington  (corrosive  sublimate,  0.8;  commercial  HC1,  60.0  c  c.;  alcohol,  400.0 
c.c.;  water,  to  IOOG.O  c.c.). 

A  very  simple  method  of  making  a  disinfectant  similar  to  lysol  is 
to  put  one  part  of  cresol  or  crude  carbolic  acid  and  one  part  of  soft  soap 
in  a  wide-mouthed  bottle  over  night.  The  resulting  compound  makes 
a  perfect  solution  with  water  and  a  5%  solution  of  this  will  be  found 
at  least  equal  to  a  5%  phenol  solution.  In  addition  to  using  as  a 
desk  jar  disinfectant  it  is  excellent  for  disinfecting  faeces,  sputum,  etc. 

For  use  in  making  loops  and  needles,  platinum  wire  of  26  gauge  will  be  found 
most  suitable.  The  handle  made  of  glass  rod  is  preferable  to  the  metal  ones.  One 
end  is  fused  in  the  flame  and,  holding  the  3-  to  4-in.  piece  of  platinum  wire,  with 
forceps,  in  the  same  flame,  insert  the  glowing  metal  into  the  molten  glass. 

For  making  smears  from  faeces,  sputum,  and  the  like,  wooden  tooth-picks  are 
very  convenient;  the  kind  with  the  spatulate  end  is  preferable. 

When  gas  is  obtainable,  the  maintaining  of  a  constant  temperature  for  the 
body  temperature  incubator  (38°  C.)  and  the  paraffin  oven  (60°  C.)  is  best  secured 
by  the  use  of  some  of  the  various  types  of  thermo-regulators.  The  Reichert  type 
is  the  one  in  general  use,  although  there  are  many  features  about  the  Dunham  and 
Roux  regulators  which  are  advantageous. 

If  the  pressure  of  the  gas-supply  varies  from  time  to  time,  it  is  essential  to  regu- 
late this  by  the  use  of  a  gas-pressure  regulator  (Murrill's  is  a  cheap  and  satisfactory 
one). 

Incubators,  controlled  electrically,  can  be  obtained  of  certain  foreign  makers, 
and  are  quoted  in  catalogues  of  American  dealers.  It  is  probable  that  the  Koch 
petroleum  lamp  incubator  is  the  most  satisafctory  one  where  gas  is  not  obtainable. 
They  should  be  of  all  metal  construction,  and  not  with  a  wood  casing,  on  account  of 
the  danger  from  fire.  They  cost  from  twenty-five  to  fifty  dollars. 

An  incubator  may  be  extemporized  by  putting  the  bulb  of  an  incandescent 
electric  lamp  in  a  vessel  of  water.  The  proper  temperature  may  be  obtained  by 
increasing  the  amount  of  water  or  by  covering  the  opening  more  or  less  completely 
with  a  towel.  The  test-tubes  to  be  incubated  can  be  put  into  a  fruit  jar  or  tin  can, 
which  receptacle  is  placed  in  the  vessel  heated  by  the  lamp. 

Emery  suggests  the  use  of  a  Thermos  bottle  as  an  incubator. 

The  vacuum  bottle  should  be  first  warmed  by  pouring  in  warm  water.  After- 
ward the  bottle  should  be  three-fourths  filled  with  water  at  100°  F. 


CAPILLARY  BULB   PIPETTES  13 

Schrup  suspends  his  cultures  and  thermometer  in  the  water  by  threads  attached 
to  pins  in  the  cork  of  the  vacuum  bottle.  The  plug  should  be  paraffined  or  covered 
with  a  rubber  cap.  As  regards  the  matter  of  a  low-temperature  incubator  (for 
gelatin  work),  this  is  best  met  by  using  a  small  refrigerator.  The  ice  in  the  upper 
part  maintains  an  even  cold,  and  by  connecting  up  an  electric  lamp  in  the  lower  part 
of  the  refrigerator  we  can  easily  maintain  a  temperature  which  only  varies  one  or 
two  degrees  during  the  twenty-four  hours. 

With  a  i6-candle-power  lamp  a  temperature  of  about  25°  C.  is  maintained  (this 
is  too  high,  being  about  the  melting-point  of  gelatin) ;  with  an  8-candle-power,  one 


10. 


FIG.  6. — i,  2,  3,  Drawing  out  glass  tubing;  4,  5,  Wright's  rubber  bulb  capillary 
pipettes  showing  grease  pencil  mark  for  making  dilutions;  6,  7,  Wright's  U-tubes; 
8,  9,  10,  methods  of  drawing  out  test-tubes  for  vaccines  in  opsonic  work;  n,  bac- 
teriological pipette. 

about  21°  to  23°  C. ;  and  with  a  4-candle-power,  from  18°  to  20°  C. ;  the  box  being 
about  20X30X36  inches. 

When  much  serum  reaction  work  is  done,  an  electrically  run  centrifuge  is  a  great 
convenience. 

A  filter  pump  attached  to  the  water  faucet,  preferably  by  screw  threads,  is 
almost  indispensable  for  filtering  cultures,  etc.,  and  for  cleaning  small  pipettes, 
especially  the  hsemocytometer  pipettes.  Such  a  filter  or  vacuum  pump  with  a 
vacuum  gauge  is  more  easily  controlled. 

The  niter  pump  is  indispensable  when  using  the  various  types  of  porcelain  or 
Berkefeld  niters.  The  Punkal  or  Muencke  types  of  filter  are  the  most  convenient  in 


14  APPARATUS 

filtering  toxins  or  in  the  sterilization   of  certain   media  when  heating  would   be 
unadvisable. 

With  the  possible  exception  of  the  platinum  loop,  there  is  no  piece 
of  apparatus  so  applicable  to  many  uses  as  the  capillary  pipette  made 
from  a  piece  of  glass  tubing. 

These  may  be  made  in  a  great  variety  of  shapes.  The  one  with  a 
hooked  end,  the  Wright  tube,  is  the  best  apparatus  for  securing  blood 


-L-AVERY- 


FIG.  7. — i,  Apparatus  combining  various  methods  for  culture  of  anaerobes;  (a) 
Hofmann  clamp  for  connecting  with  vacuum  pump;  (b)  pyrogallic  at  bottom  of 
bottle  for  Buchner's  O  absorption  method;  (c)  deep  glucose  agar  stab  covered  with 
sterile  liquid  petrolatum  (see  anaerobes).  2,  One-fourth  inch  capillary  loop  U  tube 
for  making  two  nitric  acid  albumin  tests  (see  chemical  examination  of  urine).  3, 
Piece  of  tubing  bent  to  hold  slide  for  steaming  smears  in  flame.  4,  Schmidt's  fer- 
mentation apparatus,  as  modified  by  using  graduated  cylinder  (see  under  faeces). 
5,  One-fourth  inch  glass  tubing,  4  1/2  inches  long  with  corks  at  each  end.  For 
contrifuging  faeces  for  ova.  6a,  Apparatus  connected  with  sterile  centrifuge 
tube  for  taking  blood  from  vein  of  man  or  a  guinea-pig  or  rabbit's  heart.  6b, 
Erlenmeyer  flask  which  can  be  used  instead  of  centrifuge  tube.  See  under  sections 
Immunity  and  Blood.  7,  A  graduated  pipette  with  Hofmann  clamp  applied  to 
rubber  bulb  for  precise  delivery  of  measured  quantities  of  liquids. 

for  serum  tests.  The  crook  hangs  on  the  centrifuge  guard  and  by 
filing  and  breaking  the  thicker  part  of  the  tube  the  serum  is  accessible 
to  a  capillary  rubber  bulb  pipette  or  to  the  tip  of  a  haemocytometer 
pipette.  In  this  way  dilutions  of  serum  are  easily  made.  The  capil- 


BACTERIOLOGICAL   PIPETTES  15 

lary  pipette  is  made  by  taking  a  piece  of  i/4-in.  soft  German  glass  tub- 
ing, about  6  inches  long,  and  heating  in  the  middle  in  a  Bunsen  flame, 
revolving  the  tubing  while  heating  it.  When  it  becomes  soft  in  the 
center,  remove  from  the  flame  and  with  a  steady  even  pull  separate  the 
two  ends.  The  capillary  portion  should  be  from  18  to  20  inches  in 
length.  When  cool,  file  and  break  off  this  capillary  portion  in  the 
middle.  We  then  have  two  capillary  pipettes.  By  using  a  rubber 
bulb,  such  as  comes  on  medicine  droppers,  we  have  a  means  of  sucking 
up  and  forcing  out  fluids  by  pressure  with  the  thumb  and  forefinger  of 
the  right  hand.  The  bulb  should  be  pushed  on  about  1/2  to  3/4  in.; 
this  gives  a  firmer  surface  to  control  the  pressure  on  the  bulb. 

A  bacteriological  pipette  is  made  by  drawing  out  a  g-inch  piece  of  tubing  about 
3  inches  at  either  end,  then  heating  in  the  middle  we  draw  out  and  have  two  pipettes 
similar  to  the  one  shown  in  the  drawing.  A  piece  of  cotton  is  loosely  pushed  in 
just  above  the  narrow  portion.  These  may  be  wrapped  in  paper  and  sterilized  for 
future  use.  They  may  be  made  perfectly  sterile  at  the  time  of  drawing  out. 

Where  gas  is  not  at  hand,  the  Barthel  alcohol  lamp  gives  a  flame  similar  to  that 
of  the  Bunsen  lamp  and  is  equally  satisfactory  for  heating  glass  tubing.  By  making 
a  collar  with  a  lateral  opening  to  fit  the  burner  of  a  Primus  lamp  a  powerful  side- 
flame  is  obtained  which  is  almost  as  suitable  for  glass  blowing  as  the  Bunsen  blast 
usually  employed. 


CHAPTER  II. 
CULTURE  MEDIA. 

WHILE  there  are  certain  advantages  in  sterilizing  the  glass  test- 
tubes  prior  to  filling  them  with  media,  yet  this  may  be  dispensed  with — 
the  sterilization  after  the  media  has  been  tubed  being  sufficient.  If  a 
dressing  sterilizer  is  at  hand,  this  is  preferable  for  sterilizing  such  media 
as  bouillon,  potato,  and  agar  (10  to  15  pounds'  pressure  for  fifteen  min- 
utes). Milk  should  be  sterilized  with  the  Arnold,  subjecting  the  media 
to  three  steamings  for  twenty  minutes  on  three  successive  days.  Gelatin 
may  be  sterilized  in  either  way,  but  preferably  in  the  autoclave  at  7 
pounds'  pressure  for  fifteen  minutes.  As  soon  as  taken  out  of  the  ster- 
ilizer it  should  be  cooled  as  quickly  as  possible  in  cold  water.  This 
procedure  tends  to  prevent  the  lowering  of  the  melting-point  of  the  fin- 
ished gelatin  and  also  preserves  its  spissitude. 

Blood-serum  is  preferably  solidified  as  slants  in  a  blood-serum  inspissator.  This 
requires  one  to  two  hours.  The  subsequent  sterilization  in  the  autoclave  or  Arnold 
should  not  be  done  immediately  after  making  the  solidified  slants,  but  on  the  subse- 
quent day.  If  done  on  the  same  day,  many  of  the  slants  are  ruined  by  being  dis- 
rupted by  bubbles.  The  preparation  of  blood-serum  slants  or  slants  of  egg  media 
can  be  conveniently  carried  out  in  a  rice  cooker  (double  boiler).  Place  the  tubes 
in  the  inner  compartment  of  the  cooker,  obtaining  the  slant  desired  B"y  manipulating 
an  empty  test-tube,  or  with  a  towel  or  cotton  batting  on  the  bottom.  Then  cover 
the  tubes  with  another  towel.  The  outer  compartment  should  contain  water  alone 
(no  25%  salt  solution).  The  inner  compartment  should  be  weighted  down  so  that 
it  is  surrounded  by  water — the  light  tubes  not  being  sufficient  to  sink  it.  Allowing 
the  water  in  the  outer  compartment  to  boil  one  or  two  hours  will  inspissate  or  solidify 
the  slants  satisfactorily.  The  sterilization  on  subsequent  days  may  be  carried  out 
in  the  same  apparatus,  although  it  is  more  efficient  if  done  in  an  Arnold  or  an  auto- 
clave. (This  sterilization  in  the  rice  cooker  makes  the  media  too  dry.) 

In  making  media  a  rice  cooker  is  almost  essential;  at  any  rate,  it  is  so  if  ease, 
expedition,  and  unfailing  success  in  preparation  are  to  be  achieved.  As  it  is  neces- 
sary to  make  the  contents  of  the  inner  compartment  boil,  the  temperature  of  the 
water  in  the  outer  compartment  must  be  raised.  This  is  done  by  using  a  25% 
solution  of  common  salt  or  a  20%  solution  of  calcium  chloride  in  the  outer  compart- 
ment instead  of  plain  water.  Should  CaCla  be  carried  over  to  media  in  inner  com- 
partment (as  by  thermometer)  coagulation  of  albumin  and  clearing  of  medium  will 
be  prevented. 

16 


NUTRIENT  BOUILLON  17 

A  15%  solution  of  salt  raises  the  boiling-point  2  1/2°  C.;  a  20%,  3  1/2°  C., 
and  a  25%,  4  1/2°  C.  The  raising  of  the  boiling-point  by  calcium  chloride  is  about 
the  same  for  similar  strength  solutions. 

Although  the  Bacteriological  Committee  of  the  A  P.  H.  Association  recom- 
mends special  steps  to  be  taken  in  the  preparation  of  gelatin  and  agar,  yet  for  clinical 
purposes  it  will  be  found  satisfactory  to  keep  on  hand  a  stock  of  bouillon,  and  when 
it  is  desired  to  make  agar  or  gelatin  to  simply  prepare  such  media  from  the  stock 
bouillon  in  the  way  to  be  subsequently  given. 

NUTRIENT  BOUILLON. 

This  may  be  made  either  from  fresh  meat  or  from  meat  extract. 
Media  from  fresh  meat  are  usually  lighter  in  color  and  possibly  clearer. 
In  the  Philippines,  however,  certain  measures  employed  for  the  preser- 
vation of  the  meat  made  it  very  difficult  to  prepare  clear  bouillon  from 
it,  so  that  meat  extract  was  used  entirely.  There  is  very  little  differ- 
ence, if  any,  in  the  nutritive  power  of  media  made  in  either  way.  The 
chief  objections  to  fresh  meat  as  a  base  are:  i.  It  takes  more  time  and 
trouble.  2.  The  reaction,  due  to  sarcolactic  acid  and  acid  salts,  is 
quite  acid,  so  that  it  is  necessary  to  titrate  and  neutralize  the  excess  of 
acidity.  3.  The  reaction  of  the  finished  media  tends  to  change  unless 
the  boiling  at  the  time  of  making  was  very  prolonged.  4.  It  is  not 
infrequent  to  have  a  heavy  precipitate  of  phosphates  thrown  down  at 
the  time  of  sterilization,  thus  making  it  necessary  to  repeat  the  process 
of  filtration  and  sterilization. 

If  fresh  meat  is  used,  take  about  500  grams  (one  pound),  remove  fat  and  cut  it 
up  with  a  sausage  mill  or  purchase  the  meat  already  cut  up  as  for  a  Hamburg  steak. 
It  makes  little  difference  whether  the  amount  be  100  grams  more  or  less.  Place 
the  chopped-up  meat  in  a  receptacle  and  pour  1000  c.c.  of  water  over  it.  Keep  in 
the  ice  chest  over  night  and  the  next  morning  skim  off  with  a  piece  of  absorbent  cot- 
ton the  scum  of  fat;  then  squeeze  out  the  infusion  with  a  strong  muslin  cloth,  mak- 
ing the  amount  up  to  1000  c.c.  This  meat  infusion  contains  all  the  albuminous  mate- 
rial necessary  for  the  clarification  of  the  bouillon.  It  is  convenient  to  designate  this 
meat  base  as  Meat  Infusion  to  distinguish  from  the  base  containing  meat  extract. 

Having  obtained  1000  c.c.  of  this  50%  meat  infusion,  we  dissolve  in  it  i%  of 
Witte's  peptone  and  1/2%  of  sodium  chloride.  While  there  is  a  sufficiency  of  the 
various  salts  necessary  for  bacterial  development  in  the  meat  juices,  yet  there  is  not 
enough  to  give  the  best  results  when  bouillon  cultures  of  various  organisms  are  used 
for  agglutination  tests;  and  furthermore,  when  bouillon  is  used  for  blood  cultures, 
disintegration  of  the  red  cells,  with  clouding  of  the  clear  medium,  may  occur  if  there 
be  not  sufficient  salt  present  to  prevent  this. 

The  salt  and  the  peptone  are  best  put  in  a  mortar,  and  adding  about  one  ounce 
of  the  meat  infusion  we  make  a  pasty  mass;  then  we  gradually  add  the  remaining 
infusion  until  solution  is  complete.  It  is  sometimes  recommended  to  use  a  temper- 


1 8  CULTURE    MEDIA 

ature  of  50°  C.  to  facilitate  the  solution  of  the  peptone.  This  is  not  necessary,  and 
if  the  temperature  is  not  watched  closely  it  might  go  up  to  65°  C.  or  higher  and  we 
should  lose  the  clearing  albuminous  material  from  its  coagulation.  Of  this  rather 
cloudy  solution  take  up  10  c.c.  with  a  pipette  and  let  it  run  out  into  a  porcelain 
dish.  Add  40  c.c.  of  distilled  or  rain  water  and  about  six  drops  of  a  0.5%  phenol- 
phthalein  solution.  (Phenolphthalein,  o.  5 ;  dilute  alcohol,  TOO  c.  c. )  Bring  the  con- 
tents of  the  porcelain  dish  to  a  boil  and  continue  boiling  for  one  or  two  minutes  in 
order  to  expel  all  CC>2.  Now  from  a  burette  filled  with  decinormal  sodium  hydrate 
solution,  run  in  this  solution  until  we  have  the  development  of  a  faint  but  distinct 
pink  in  the  boiling  diluted  bouillon  which  is  not  dissipated  on  further  boiling. 

It  is  more  satisfactory  to  take  burner  from  beneath  the  porcelain  dish  just  before 
running  in  the  N/io  solution,  again  boiling  so  soon  as  a  pink  color  is  obtained. 
Having  obtained  the  light  pink  coloration  we  read  off  the  number  of  c.c.  or  frac- 
tions of  a  c.c. of  N  /io  sodium  hydrate  solution  added  to  produce  the  color.  This 
number  gives  the  acidity  of  the  bouillon  in  percentage  of  N/i  acid  solution. 

Percent  acid  means  that  so  many  c.c.  of  N/i  acid  added  to  100  c.c.  of  the  medium 
at  the  neutral  point  would  give  that  percentage  reaction.  Thus  11/2  c.c.  of  N/i 
HC1  solution  added  to  100  c  c.  of  medium  at  o,  would  give  us  i  1/2%  of  acidity 
or  +1.5. 

Percent  alkaline  means  so  many  c.c.  of  N/i  sodium  hydrate  solution  added  to 
100  c.c.  of  the  medium  at  the  neutral  point.  Thus  a  1/2%  alkaline  medium  would 
be  one  whose  alkalinity  would  correspond  to  the  addition  of  1/2  c.c.  of  N/i 
NaOH  to  100  c.c.  of  the  medium  at  o.  It  is  written  —0.5. 

If  we  took  100  c.c.  of  the  medium  and  put  it  in  a  beaker  and  then  ran  in  N/i 
NaOH  solution  from  a  burette,  it  will  be  readily  understood  that  if  we  had  to  add 
3  1/2  c.c.  of  N/i  NaOH  to  obtain  the  pink  color,  it  would  show  that  the  acidity  of 
the  100  c.c.  of  medium,  being  tested,  corresponded  to  3.5  c.c.  of  N/i  acid  solution, 
and  that  its  acidity  was  equal  to  3  1/2%  of  N/i  acid  solution,  or  that  its  reaction 
was  +3.5. 

As  N/i  NaOH  solution  is  too  corrosive  for  general  use  in  a  burette,  and  as  io  c.c. 
of  medium  is  more  convenient  to  work  with  than  100  c.c.,  we  use  a  solution  one- 
tenth  the  strength  of  the  N/i  NaOH  and  we  take  only  one-tenth  of  the  100  c.c.  of 
medium.  In  this  way  it  is  the  same  from  a  standpoint  of  directly  reading  off  our 
percentage  reaction  as  if  we  had  100  c.-c.  of  medium  and  used  N/i  NaOH  solution. 
The  A.  P.  H.  Association  recommends  5  c.c.  of  the  medium  and  the  use  of  N/2O 
NaOH.  As  the  N/io  NaOH  is  always  at  hand  for  titrating  gastric  juice,  the  N/io 
is  used  instead. 

Should  it  be  found  difficult  to  carry  on  the  titration  while  boiling  the  end  reaction 
may  be  fairly  accurately  determined  in  the  cold.  Deliver  into  a  beaker  from  a 
pipette  io  c.c.  of  the  bouillon  and  make  up  to  50  c.c.  with  distilled  water  and  add 
5  drops  of  o.  5%  phenolphthalein  solution.  Then  run  in  N/io  NaOH  from  a  burette 
and  continue  to  add  the  N/io  NaOH  solution  from  the  burette,  drop 
by  drop,  until  the  addition  of  a  drop  fails  to  show  any  intensifying  of  the  purplish 
violet  color  at  the  spot  where  it  came  in  contact  with  the  diluted  bouillon  in  the 
beaker.  This  marks  the  end  reaction.  A  reaction  of  about  +o.  7  in  the  cold  gives  a 
delicate  pink  with  phenolphthalein  as  an  indicator.  Titration  in  the  cold  is  not  very 
satisfactory  with  gelatin  and  agar. 


TITRATION    OF   MEDIA  1 9 

Having  determined  the  percentage  acidity 'of  the  10  c.c.  sample  tested,  we  easily 
calculate  the  number  of  c.c.  of  N/i  NaOH  solution  required  to  be  added  to  the  1000 
c.c.  of  bouillon  to  obtain  a  reaction  corresponding  to  the  neutral  point  of  phenol- 
phthalein.  It  is  more  exact  to  take  the  average  of  two  titrations. 

As  100  c.c.  of  medium  would  require  3  1/2  c.c.,  1000  c.c.  would  require  10  times 
as  much,  or  35  c.c.  N/i  NaOH  solution.  Having  measured  out  and  added  35  c.c 
of  the  N/i  NaOH  solution  to  the  meat  infusion,  containing  salt  and  peptone,  we 
have  a  solution  which  is  exactly  neutral  to  phenolphthalein,  or  o.  It  is  usually 
considered  that  a  reaction  of  about  i%  acid  is  the  optimum  reaction  for  bacterial 
growth.  Hence  we  should  now  add  i%  of  N/i  HC1  solution  to  the  medium.  This 
would  be  accomplished  by  adding  10  c.c.  of  N/i  HC1  solution  to  the  1000  c.c.  of 
neutralized  medium,  and  we  would  have  a  medium  with  a  reaction  of  +i.  If  we 
desired  a  reaction  of  i%  alkalinity  we  would  add  an  additional  c.c.  of  N/i  NaOH 
solution  to  every  100  c.c.  of  the  medium  at  o,  or  10  c.c.  for  the  1000  c.c.  of  medium. 
The  reaction  would  then  be  —  i. 

As  a  matter  of  convenience,  we  usually  determine  the  reaction  of  the  medium, 
which  is  always  more  or  less  acid,  and  then  add  enough  N/i  NaOH  to  reduce  the 
acidity  to  the  percentage  we  desire  to  set  the  medium,  instead  of  neutralizing  all 
the  acidity  present  and  then,  in  a  second  operation,  restoring  the  acidity  to  the 
point  desired. 

Thus  finding  the  acidity  of  the  medium  to  be  3  1/2%  and  desiring  to  give  it 
an  acidity  of  i%,  we  would  add  only  2  1/2  c.c.  of  N/i  NaOH  to  every  100  c.c.  of 
medium,  or  25  c.c.  for  the  zoooc.c.  of  medium.  The  reaction  would  then  be  found 
to  be  +i. 

The  neutral  point  of  litmus  is  not  a  sharp  one,  but  it  corresponds  rather  closely 
with  a  reaction  of  +1.5  to  phenolphthalein.  The  recommendations  of  the  A.  P.  H. 
Association  call  for  making  the  titration  with  the  medium  boiling.  If  the  color  of 
the  end  reaction  at  boiling-point  be  obtained,  it  will  be  found  that  when  cool  it 
deepens  until  it  corresponds  to  the  rich  violet-pink  of  the  end  reaction  in  the  cold 
or  vice  versa. 

To  summarize: 

Take  Peptone,  10  grams 

Sodium  chloride,  5  grams 

50%  meat  infusion,  1000  c.c. 

Dissolve  the  peptone  and  sodium  chloride  in  the  meat  infusion  and 
add  enough  N/i  NaOH  to  make  the  reaction  +i. 

Put  the  solution  in  the  inner  compartment  of  a  rice  cooker  and  bring 
to  the  boiling-point  and  maintain  this  temperature  for  twenty  minutes. 
The  calcium  chloride  or  sodium  chloride  in  the  outer  compartment  of 
the  rice  cooker  enables  us  to  secure  a  boiling  temperature  for  the  con- 
tents of  the  inner  compartment.  Do  not  stir  the  bouillon  that  is  being 
heated,  as  the  pultaceous  membranous  mass  of  coagulated  albumin 
makes  nitration  easy.  Filter.  The  filter-paper  in  the  funnel  should  be 


20  CULTURE    MEDIA 

thoroughly  wet  with  water  before  pouring  on  the  bouillon.  This  is  to 
prevent  clogging  of  the  pores  of  the  filter-paper.  Make  up  the  quantity 
of  filtrate  to  1000  c.c.  by  adding  water. 

If  greater  exactness  is  demanded  than  answers  for  ordinary  clinical  work,  it  is 
advisable  to  again  titrate  and  again  adjust  the  reaction  or  to  simply  record  the  exact 
reaction.  It  is  more  convenient  to  have  a  counterpoise  to  balance  the  inner  compart- 
ment and  then  to  add  water  to  the  medium  until  a  kilo  weight,  in  addition  to  the 
weight  balancing  the  container,  is  just  balanced.  Then  titrate,  adjust  the  reaction 
(if  so  desired),  and  filter.  Sterilize  in  the  autoclave  at  115°  C.  for  fifteen  minutes 
or  in  the  Arnold  on  three  successive  days.  The  use  of  a  balance  is  preferable  in  the 
preparation  of  bouillon,  necessary  in  making  gelatin  and  imperative  in  making 
agar  media. 

BOUILLON  MADE  FROM  LIEBIG'S  MEAT  EXTRACT. 

Place  in  a  mortar  3  grams  of  Liebig's  extract,  10  grams  of  peptone  and  5  grams 
of  sodium  chloride.  Dissolve  the  whites  of  one  or  two  eggs  in  1000  c.c.  of  water. 
Then  add  this  egg-white  water,  little  by  little,  to  the  extract,  peptone,  and 
salt  in  the  mortar  until  a  brownish  solution  is  obtained.  Pour  this  into  the  inner 
compartment  of  a  rice  cooker;  apply  heat  to  the  outer  compartment  containing 
the  salt  or  calcium  chloride  solution,  allow  to  come  to  a  boil  and  to  continue 
boiling  for  fifteen  to  twenty  minutes.  Do  not  stir.  Place  inner  compart  ment  on 
the  scales  and  its  counterpoise  and  a  one-kilo  weight  on  the  other  side.  Add 
water  until  the  two  arms  balance.  Filter  and  sterilize. 

The  reaction  of  media  made  with  Liebig's  meat  extract  rarely  exceeds  +0.75 
(from  +0.6  to  +0.9).  Consequently  for  growing  bacteria  it  is  unnecessary  to  titrate 
and  adjust  reactions  unless  precision  is  demanded. 

SUGAR-FREE  BOUILLON. 

Inoculate  nutrient  bouillon  in  a  flask  with  the  colon  bacillus.  Allow  to  incubate 
at  37°  C.  over  night.  Pour  the  contents  into  a  sauce-pan  and  bring  to  a  boil  to  kill 
the  colon  bacilli.  Put  about  15  grams  of  purified  talc  (Talcum  purificatum,  U.  S.  P. ) 
in  a  mortar.  Add  the  dead  colon  culture,  stirring  constantly.  Then  filter  through 
filter-paper.  It  may  be  necessary  to  again  pass  the  filtrate  through  the  same 
filter  until  the  sugar-free  bouillon  is  perfectly  clear. 

For  all  ordinary  purposes  the  very  small  amount  of  sugar  in  bouillon  made  from 
Liebig's  meat  extract  may  be  neglected  in  determining  gas  production;  so  that  under 
such  conditions  the  various  sugars  could  be  added  directly  to  the  meat-extract 
bouillon. 

SUGAR  BOUILLONS. 

The  sugar  media  ordinarily  used  for  determining  fermentation  or  gas  production 
are  those  of  glucose  and  lactose.  In  special  work  such  carbohydrates  as  saccharose 
and  maltose  are  used.  The  alcohol  mannite  is  used  in  differentiating  strains  of 
dysentery  bacilli. 


PEPTONE    SOLUTION  21 

To  make,  simply  dissolve  i  or  2%  of  the  sugar  in  sugar-free  bouillon  or  that 
made  from  meat  extract.  Tube  in  Durham's  or  the  ordinary  fermentation  tubes  and 
sterilize  in  the  autoclave  at  only  about  5  pounds'  pressure  for  15  minutes,  or  in  the 
Arnold.  Ordinary  peptone  solution  is  a  good  substitute  for  sugar-free  bouillon. 

Too  high  a  degree  of  heat  may  turn  the  sugar  bouillon  brownish.  The  nature 
of  the  sugar  itself  ma}7  further  be  affected  by  too  high  a  temperature. 

CALCIUM  CARBONATE  BOUILLON. 

Where  we  wish  to  cultivate  such  organisms  as  streptococci  and  pneumococci  in 
massive  cultures  we  may  add  small  fragments  of  marble  (calcium  carbonate)  so 
that  any  inimical  excess  of  acid  may  be  neutralized.  North  used  a  glucose  bouillon 
containing  calcium  carbonate  in  the  production  of  massive  cultures  of  B.  bulgaricus. 

GLYCERINE  BOUILLON. 

Add  6%  of  glycerine  to  ordinary  bouillon.  It  is  chiefly  used  in  the  cultivation 
of  tubercle  bacilli. 

PEPTONE  SOLUTION  (DUNHAM'S). 

Dissolve  i%  of  Witte's  peptone  and  1/2%  of  sodium  chloride  in  distilled  water. 
Filter,  tube,  and  sterilize.  Peptone  solution  may  be  used  as  a  base  for  sugar  media 
instead  of  bouillon.  It  is  the  medium  used  in  testing  for  indol  production.  This 
test  is  made  by  adding  from  6  to  8  drops  of  concentrated  H2SO4  to  a  twenty-four-  to 
forty-eight-hour-old  peptone  culture  of  the  organism  to  be  tested.  If  the  organism 
produces  both  indol  and  a  nitroso  body,  we  obtain  a  violet-pink  coloration,  "cholera 
red. "  If  no  pink  color  is  produced  on  the  addition  of  the  sulphuric  acid,  add  about 
i  c.c.  of  an  exceedingly  dilute  solution  (i  :  10,000)  of  sodium  nitrite. 

It  is  very  important  in  determining  the  "cholera  red"  reaction  to  know  that  the 
peptone  used  will  give  the  reaction  as  it  is  not  given  by  true  cholera  strains  with 
certain  samples  of  peptone. 

For  the  Voges-Proskauer  Reaction. — Fill  fermentation  tubes  with  a  2%  glucose 
Dunham's  peptone  solution  and  sterilize.  After  inoculation  with  the  organism  to 
be  tested  incubate  for  three  days.  Then  add  2  to  3  c.c.  of  strong  caustic  potash 
solution.  The  development  of  a  pink  color  on  exposure  to  the  air  is  a  positive 
reaction  (the  color  of  a  weak  eosin  solution). 

Hiss'  SERUM  WATER  MEDIUM. 

Take  one  part  of  clear  beef  serum  and  add  to  it  about  3  times  it's  bulk  of  water. 
Heat  the  mixture  in  the  Arnold  for  15  minutes  to  destroy  any  diastatic  ferment  which 
might  be  present.  Color  to  a  deep  transparent  blue  with  litmus  solution  and  then 
add  i%  of  any  of  the  various  sugars  used  in  fermentation  tests.  Sterilize  in 
the  Arnold  by  the  fractional  method. 

NUTRIENT  AGAR. 

In  making  agar  medium  it  is  preferable  to  use  powdered  agar,  as  this  goes  into 
solution  more  readily  than  the  shredded  agar.  The  reaction  of  agar  is  slightly 


22  CULTURE    MEDIA 

alkaline,  so  that  if  i  1/2  to  2%  of  agar  is  added  to  nutrient  bouillon  having  a  reaction 
of  +i  the  finished  product  will  be  found  to  be  about  +0.8. 

To  make:  Weight  out  15  to  20  grams  of  powdered  agar  and  place  in  a  mortar. 
Make  a  paste  by  adding  nutrient  bouillon,  little  by  little,  and  when  a  smooth  even 
mixture  is  made,  pour  in  into  the  inner  compartment  of  a  rice  cooker  and  add  the 
remainder  of  the  1000  c.c  of  bouillon.  The  use  of  the  balance  is  preferable. 

The  outer  compartment  of  the  rice  cooker  should  contain  the  25%  salt  solution. 
Bring  to  boil,  and  the  agar  will  be  found  to  have  entirely  gone  into  solution  after 
five  to  ten  minutes  of  boiling. 

Then,  using  a  funnel  which  has  been  heated  in  boiling  water  and  which  contains 
a  small  pledget  of  absorbent  cotton,  we  filter  the  agar,  tube  it,  and  sterilize  it  in  the 
autoclave  or  Arnold.  One  and  one-half  percent  agar  can  be  readily  filtered  through 
filter-paper  and  gives  a  clearer  medium. 

By  taking  of  meat  extract  3  grams,  peptone  10  grams,  salt  5  grams,  powdered 
agar  15  grams,  the  white  of  one  egg  and  1000  c.c.  of  water,  making  at  first  apasteof 
all  the  ingredients  in  a  mortar,  then  gradually  adding  the  remainder  of  the  1000  c.c. 
of  water,  putting  in  the  rice  cooker,  bringing  to  a  boil  without  stirring,  allowing  to 
boil  fifteen  minutes  and  then  filtering  through  absorbent  cotton  placed  between  two 
layers  of  gauze  in  a  hot  funnel,  we  obtain  a  satisfactory  medium,  the  reaction  of 
which  will  be  from  -fo.  7  to  +0.9.  It  is  very  important  not  to  interfere  with  the  pul- 
taceous  coagulum  which  forms  on  the  surface  of  the  boiling  agar. 

Where  very  exact  adjustment  of  the  reaction  of  the  finished  pro- 
duct is  desirable  the  method  of  preparation  of  the  Committee  on 
Water  Analysis  of  the  American  Public  Health  Association  is  to  be 
preferred. 

Dissolve  15  grams  of  agar  in  500  c.c.  of  water  in  the  inner  compartment  of  the 
rice  cooker  previously  described.  After  the  agar  is  in  solution  (after  10  to  15  minutes 
boiling)  remove  the  inner  compartment,  containing  the  3%  agar  solution,  and 
allow  it  to  cool  to  about  55°  C.  Mix  in  the  mortar,  as  described  in  the 
directions  for  making  nutrient  bouillon  from  Liebig's  extract,  3  grams  of  Liebig's 
extract,  10  grams  of  peptone  and  5  grams  of  sodium  chloride  in  500  c.c.  of  water 
containing  the  whites  of  one  or  two  eggs.  Heat  this  mixture  to  50  to  55°  C.  and  pour 
it  into  the  agar  solution,  in  the  inner  compartment,  which  has  been  cooled  to  about 
55°  C.  Now  titrate  this  mixture  containing  500  c.c.  of  double  strength  agar  and  500 
c.c.  of  double  strength  peptone,  meat  extract  and  salt  solution.  The  resulting 
1000  c.c.  gives  i  1/2%  agar  and  i%  peptone  solution.  Having  adjusted  the 
reaction  by  the  addition  of  the  necessary  amount  of  N/i  acid  or  alkali,  we  place  the 
inner  compartment  in  the  outer  one  of  the  rice  cooker,  bring  to  a  boil  and  filter 
through  filter-paper  which  has  been  wetted  with  boiling  water.  The  filtration  can 
be  carried  out  in  the  autoclave  or  in  an  Arnold  sterilizer.  Of  course  the  ordinary 
filtering  through  gauze  and  cotton  will  answer  where  clearer  media  is  not  an  object. 

GLUCOSE  AGAR. 

Add  the  agar  to  i  or  2%  glucose  bouillon  and  proceed  as  for  ordinary  agar.  If 
preferred,  the  glucose  agar  can  be  made  by  rubbing  up  meat  extract  3  grams,  peptone 


GELATIN  23 

10  grams,  salt  5  grams,  glucose  10  grams  and  15  grams  of  agar  in  1000  c.c.  of  water 
containing  the  white  of  egg  (one  to  two  eggs),  then  boiling  in  the  rice  cooker  and 
filtering. 

GLYCERINE  AGAR. 

Add  the  agar  to  6%  glycerine  bouillon  instead  of  nutrient  bouillon,  or  the  gly- 
cerine may  be  added  to  nutrient  agar  which  has  been  melted.  Glycerine  agar  with 
a  reaction  of  o  makes  an  excellent  base  for  blood  and  serum  media  for  use  in  cul- 
turing  delicate  pathogens. 

GLYCERINE  AGAR  EGG  MEDIUM. 

Take  the  white  and  the  yolk  of  one  egg  and  mix  thoroughly  in  a  vessel  kept 
between  45°  and  55°  C.  with  an  equal  amount  of  glycerine  agar.  Tube  the  medium, 
inspissate  in  a  rice  cooker  as  for  serum  tubes,  and  sterilize  as  for  blood-serum  tubes. 

This  makes  an  excellent  medium  for  growing  tubercle  bacilli.  As  egg  medium 
has  a  tendency  to  be  dry,  it  is  well  to  add  i  c.c.  of  glycerine  bouillon  to  each  slant 
before  autoclaving. 

NUTRIENT  GELATIN. 

Place  in  a  mortar  3  grams  of  Liebig's  extract,  10  grams  of  peptone  and  5  grams 
of  sodium  chloride.  Dissolve  the  whites  of  one  or  two  eggs  in  1000  c.c.  of  water. 
Then  add  this  egg-white  water,  little  by  little,  to  the  meat  extract,  peptone  and  salt, 
in  the  mortar,  until  a  brownish  solution  is  obtained.  Pour  this  into  the  inner 
compartment  of  the  rice  cooker  and  bring  the  temperature  up  to  45°  C.  (This 
preliminary  elevation  of  temperature  is  better  carried  out  in  some  heated  water  in 
a  pan,  as  the  heating  by  means  of  the  salt  solution  in  the  outer  compartment  of  the 
rice  cooker  is  difficult  to  control,  so  that  a  temperature  approximating  70°  C.  might 
be  obtained  and  the  albumin  of  the  white  of  egg  coagulated.  The  temperature  in 
the  outer  compartment  might  be  approaching  boiling  before  the  contents  of  the  inner 
compartment  would  show  45°  C.)  Now  take  about  120  grams  of  "gold  label"  or 
other  good  quality  gelatin  (12%)  and  crush  it  down  in  the  meat  extract  egg- water 
solution  in  the  inner  compartment  of  the  rice  cooker. 

The  gelatin  quickly  goes  into  solution  at  45°  C.  Gelatin  being  quite  acid  it  will 
probably  be  found  upon  titration  that  the  reaction  is  about  +4%.  N/i  NaOH 
solution  is  added  to  bring  the  reaction  to  about  +1%.  or  3  c.c.  N/i  NaOH  for 
each  100  c.c.,  provided  the  reaction  were  exactly  +4%.  The  procedure  is  the 
same  as  for  bouillon.  The  color  reaction  is  not  quite  as  distinct  with  gelatin  as  with 
bouillon. 

Having  neutralized  and  allowed  to  boil  for  fifteen  minutes,  we  filter  through 
filter-paper  in  a  hot  funnel.  As  it  is  very  important  that  gelatin  should  be  perfectly 
clear,  it  is  better  to  filter  through  filter-paper  than  through  cotton.  The  filter-paper 
should  be  very  thoroughly  wetted  with  very  hot  water  before  filtering  gelatin  or 
agar. 


24  CULTURE    MEDIA 

Tube  the  "medium  and  sterilize,  either  in  the  Arnold  on  three  successive  days  or 
in  the  autoclave  at  8-10  pounds'  pressure  for  ten  minutes.  The  tubes  should  be 
cooled  as  quickly  as  possible  in  cold  water  after  taking  out  of  the  sterilizer. 

AGAR  GELATIN  MEDIUM  (NORTH). 

Lean  chopped  beef  or  veal,  500  grams. 

Agar,  10  grams. 

Gelatin,  Gold  label,  20  grams. 

Peptone,  Witte's,  20  grams. 

Sodium  chloride,  5  grams. 

Distilled  water,  q.s.,  1000  c.c. 

Extract  the  chopped  beef  with  500  c.c.  distilled  water  for  18  hours,  strain  through 
muslin  and  combine  the  ingredients  in  the  usual  way.  Adjust  the  reaction  to  the 
neutral  point,  using  phenolphthalein  as  indicator. 

North  states  that  this  medium  is  excellent  for  streptococci,  pneumococci  and 
diphtheria  bacilli  because  it  is  soft,  moist,  and  can  be  used  at  37°  C. 

It  is  claimed  to  be  of  special  value  for  carrying  stock  cultures. 

LITMUS  MILK. 

Milk  for  media  should  be  as  fresh  as  possible.  It  should  then  be  put  in  a  1000 
c.c.  Erlenmeyer  flask,  sterilized  for  fifteen  minutes  in  the  Arnold,  and  set  over  night 
in  the  refrigerator.  The  next  morning  the  milk  beneath  the  cream  should  be 
siphoned  off.  The  short  arm  of  the  siphon  should  not  reach  the  bottom  of  the  flask 
so  as  to  avoid  the  sediment.  Add  sufficient  litmus  solution  to  this  milk  to  give  a 
decided  lilac  tinge;  tube  and  sterilize  in  the  Arnold  on  three  successive  days. 

Litmus  milk  which  apparently  is  as  satisfactory  as  the  above  as  regards  nutritive 
quality  and  cultural  characteristics  can  be  made  from  certain  canned  milks  which 
have  not  been  condensed  or  sweetened  and  which  do  not  contain  chemical  pre- 
servatives. The  "Natura"  brand  of  milk  is  the  one  I  have  experimented  with. 

Litmus  Solution. — A  simple  solution  may  be  made  by  digesting  the  powdered 
cubes  repeatedly  with  hot  water,  mixing  the  extracts,  and,  after  allowing  them  to 
stand  all  night,  decanting  the  solution  from  the  inert  sediment  into  a  clean  bottle. 

In  litmus  solution  so  made,  however,  a  red  dye  is  also  present  while  calcium  and 
other  salts  are  dissolved  out.  For  bacteriological  purposes  a  pure  solution  of  the 
blue  dye  should  be  used.  This  is  called  "azolitmin."  It  is  freely  soluble  in  water 
but  insoluble  in  alcohol. 

It  can  be  conveniently  prepared  as  follows:  Weigh  out  2  ounces  of  powdered 
litmus;  digest  repeatedly  with  fresh  quantities  of  hot  water  until  all  the  coloring 
matter  is  dissolved  out;  allow  to  settle,  and  decant  off  the  fluid  from  the  insoluble 
powder.  Add  together  the  extracts,  which  should  measure  about  a  liter.  Evap- 
orate down  the  solution  to  a  moderate  bulk,  then  add  a  slight  excess  of  acetic  acid, 
so  as  to  convert  all  carbonates  present  into  acetates.  Continue  the  evaporation, 
the  later  stages  over  a  water  bath,  until  the  solution  becomes  pasty.  Add  200  c.c. 
of  alcohol,  and  mix  thoroughly.  The  alcohol  precipitates  the  blue  coloring  matter, 
while  a  red  coloring  matter,  together  with,  the  alkaline  acetate  present,  remains  in 


BLOOD    SERUM  25 

solution.  Transfer  to  a  filter.  Wash  out  the  dish  with  alcohol  and  add  this  to 
the  filter.  Wash  the  precipitate  on  the  filter  with  alcohol.  Dissolve  the  pure 
coloring  matter  remaining  on  the  filter  in  warm  distilled  water  and  dilute  to  500  c.c. 
Azolitmin  solution  prepared  in  this  way  is  more  sensitive  than  ordinary  litmus 
solution. 

Azolitmin  in  powder  can  be  purchased  from  dealers  in  chemicals. 

POTATO  SLANTS. 

Take  Irish  potatoes  and  scrub  throughly  with  a  stiff  brush.  Then  pare  off 
generously  all  the  outer  portion.  From  the  white  interior  cut  out  cylinders  with  a 
cork  borer.  These  cylinders  should  be  of  1/2  to  3/4  of  an  inch  in  diameter.  Di- 
vide a  cylinder  by  a  diagonal  cut.  This  gives  a  plug  with  a  flat  base,  the  other 
extremity  being  a  slant.  These  potato  plugs  should  be  left  in  running  water  over 
night  or  washed  with  frequent  changes  of  water.  This  prevents  the  blackening  of 
the  plug.  Into  a  i-in.  test-tube  drop  a  pledget  of  absorbent  cotton  well  moistened 
with  water.  Then  drop  in  the  potato  plug,  base  downward.  Sterilize  in  the  auto- 
clave at  15  pounds  for  fifteen  to  twenty  minutes,  to  insure  sterility. 

For  glycerine  potato,  soak  the  plugs  in  6%  glycerine  solution  for  about  one  hour. 
Then  drop  a  pledget  of  absorbent  cotton  moistened  with  the  same  glycerine 
solution  into  the  test-tubes  and  follow  it  with  the  potato  plug.  Sterilize  in  the 
autoclave. 

BLOOD-SERUM. 

The  blood  of  cattle  should  be  collected  in  large  pans  or  pails  at  the  abattoir. 
This  vessel  of  blood  should  then  be  kept  in  the  cold-storage  room  and  the  next 
morning  the  more  or  less  clear  serum  will  have  been  squeezed  out  from  the  clot. 
Collect  this  serum  and  keep  in  the  ice  chest  for  future  use.  If  to  be  kept  for  along 
time,  it  is  advisable  to  add  about  2%  of  chloroform  to  the  serum  in  tightly  corked 
flasks.  This  will  not  only  keep  the  serum,  but  will  eventually  sterilize  it. 

To  make  Loffler's  serum,  take  one  part  of  glucose  bouillon  and  three  parts  of 
blood-serum.  Mix,  tube,  and  coagulate  the  albumin  in  the  inspissator  or  rice  cooker, 
giving  the  tubes  a  proper  slant  before  heating.  Sterilize  the  following  day  in  the 
autoclave  as  previously  directed  (7  Ibs.)  or  in  the  Arnold  on  three  successive  days. 

A  SUBSTITUTE  FOR  ORDINARY  BLOOD-SERUM. 

Add  from  10  to  15  c.c.  of  i%  glucose  bouillon  to  the  white  and  yolk  of  one  egg, 
make  a  smooth  mixture  in  a  mortar  and  tube.  Inspissate  and  sterilize  as  for  ordin- 
ary serum  slants.  The  morphology  of  the  diphtheria  bacilli  and  the  luxuriance  of 
growth  is  similar  to  that  of  cultures  on  Loffler's  serum. 

When  this  medium  is  to  be  used  for  culturing  tubercle  bacilli  add  about  i  c.c. 
of  glycerine  bouillon  to  each  tube  before  final  sterilization  in  the  autoclave.  The 
cotton  plugs  should  be  paraffined  to  prevent  drying  of  the  slants  in  the  incubator. 
This  medium  seems  to  answer  as  a  substitute  for  Dorset's  egg  medium.  While 
glycerine  bouillon  favors  growth  of  human  tuberculosis,  it  is  not  so  satisfactory  for 


26  CULTURE    MEDIA 

bovine  tuberculosis  as  plain  glucose  bouillon.  Tim  is  better  than  the  various 
white  of  egg  substitutes  usually  recommended.  (Pouring  a  little  alcohol  in  the  mor- 
tar and  moistening  the  sides  by  tilting,  then  burning  off  the  alcohol,  in  a  measure 
sterilizes  the  mortar.  If  the  egg  is  cracked  open  with  a  sterile  knife,  a  medium  can 
be  prepared  which  will  be  sterile  as  the  result  of  the  two-hour  inspissation  in  the  rice 
cooker.)  By  covering  the  tube  with  a  rubber  cap  or  preferably,  by  heating  the  plugged 
end  of  the  test-tube,  quickly  withdrawing  the  cotton  plug  and  dipping  the  part  of 
the  plug  which  enters  the  tube  into  hot  melted  paraffin,  then  quickly  reintroducing 
the  plug,  the  contents  of  the  tube  will  be  prevented  from  drying  out.  This  procedure 
is  essential  for  growing  tubercle  bacilli.  Dorset's  egg  medium  for  the  cultivation 
of  tubercle  bacilli  consists  of  the  whole  egg,  which  is  emulsified  as  above,  and  heated 
at  70°  C.  for  from  four  to  five  hours  each  day  for  two  days.  To  provide  moisture 
about  i  c.c.  of  sterile  6%  glycerine  solution  is  added  to  each  slant. 

HYDROCELE,  AND  BLOOD  AGAR. 

To  tubes  of  melted  agar  at  50°  C,  add  from  i  to  3  c.c.  of  hydrocele  or  ascitic 
fluid,  observing  aseptic  precautions.  For  blood  agar  the  blood  from  a  vein  should 
be  received  into  a  sodium  citrate  salt  solution  to  prevent  coagulation,  and  added 
subsequently  as  for  hydrocele  fluid.  Allow  the  agar  to  solidify  as  a  slant,  or  as  a 
poured  plate. 

BLOOD-STREAKED  AGAR. 

Sterilize  the  lobe  of  the  ear  and  puncture  with  a  sterile  needle.  Collect  the  ex- 
uding blood  on  a  large  platinum  loop  and  smear  it  over  the  surface  of  an  agar  slant. 
It  is  advisable  to  incubate  over  night  as  a  test  for  sterility.  Plates  or  slants  of  glycer- 
ine agar  of  neutral  reaction  smeared  with  blood  give  the  best  results  when  such 
delicate  pathogens  as  pneumococci,  streptococci,  gonococci  or  meningococci  are  to 
be  cultured. 

BILE  MEDIA. 

Secure  ox  bile  from  the  abattoir  or  human  bile  from  cases  of  gall-bladder  drain- 
age in  hospitals.  Put  about  10  c.c.  in  each  tube  and  sterilize.  Some  prefer  to  add 
i%  of  peptone.  Conradi's  medium  is  ox  bile  containing  10%  of  glycerine  and  2% 
of  peptone.  This  is  the  medium  for  blood  cultures  in  typhoid,  etc. 

The  bile  lactose  medium  now  used  in  water  analysis  is  made  by  adding  i%  of 
lactose  to  ox  bile  and  tubing  in  fermentation  tubes.  As  a  substitute  for  fresh  bile 
one  may  use  a  15  to  20%  solution  of  a  good  quality  of  inspissated  ox  gall  (Fel  Bovis 
Purificatum).  A  liver  bouillon  made  by  using  500  grams  of  finely  divided  beef 
liver  in  1000  c.c.  of  water  with  i%  peptone,  and  prepared  as  for  meat  infusion  broth, 
is  a  good  substitute  for  bile. 

RECTOR'S  BILE  LACTOSE  NEUTRAL  RED  MEDIUM. 

This  is  recommended  in  the  isolation  of  the  colon  bacillus  as  superior  to  lactose 
litmus  agar.  It  consists  of  10%  of  dried  ox  bile,  i%  of  peptone,  and  i  1/2%  agar. 


MEDIA  27 

After  the  medium  is  filtered  and  tubed  we  add  i%  of  lactose  and  i%  of  a  i-ioo 
neutral  red  solution.  Colon  colonies  have  a  distinct  purplish  red  zone.  Furthermore 
the  bile  inhibits  the  growth  of  many  organisms  which  give  pink  colonies  on  lactose 
litmus  agar.  MacConkey's  bile  salt  medium  contains  1/2%  of  sodium  taurocholate 
and  is  colored  with  neutral  red. 

THALMAN'S  MEDIUM  FOR  THE  GONOCOCCUS. 

Five  hundred  grams  of  lean,  finely  minced  beef  are  placed  in  1000  c.c.  of  distilled 
water  and  allowed  to  stand  over  night  in  an  ice  box.  It  is  then  filtered  and  the  fil- 
trate made  up  to  1000  c.c.  with  distilled  water.  To  100  c.c.  of  the  beef  juice  add 
i  1/2  grams  of  agar,  and  boil  for  15  minutes.  Then  add  2  grams  of  glucose,  and  bring 
the  reaction  to  plus  0.6  by  addition  of  N/iNaOH.  Tube,  sterilize,  slant,  and  in- 
cubate over  night.  No  peptone  or  salt  is  required. 

PLATING  MEDIA  FOR  F^CES  WORK. 

The  media  of  Endo,  Conradi-Drigalski  and  the  lactose  litmus  agar  medium  are 
probably  the  most  satisfactory  of  the  numerous  ones  that  have  been  proposed  for 
plating  out  faeces.  A  convenient  way  of  preparing  any  one  or  all  of  these,  and  which 
apparently  gives  media  equal  to  that  prepared  according  to  the  original  formulae, 
is  as  follows: 

Liebig's  extract,  5  grams. 

Salt,  5  grams. 

Pep  ton,  10  grams. 

Agar,  30  grams. 

Water  to  make  1000  c.c. 

Prepare  as  for  ordinary  nutrient  agar,  with  the  difference  that  the  reaction  should 
be  brought  down  to  o.  Some  prefer  a  reaction  of  +0.2. 

A  stiff  agar  (3%)  is  employed  to  check  the  diffusion  of  acid  beyond  the  colony. 

FOR  ENDO'S  MEDIUM. 

Keep  this  agar  base  in  100  c.c.  quantities  in  Erlenmeyer  flasks  instead  of  test- 
tubes.  (If  more  convenient  smaller  quantities  may  be  put  in  the  flask.)  When 
needed  for  plating,  melt  a  flask  of  this  agar,  and  while  liquid  add  to  the  100  c.c. 
six  drops  of  a  saturated  alcoholic  solution  of  basic  fuchsin,  and  then  about  twenty 
drops  of  a  Treshly  prepared  10%  solution  of  sodium  sulphite.  The  sulphite  solution 
decolorizes  the  intense  red  of  the  fuchsin  to  a  light  rose  pink.  This  color  fades  to  a 
light  flesh  or  pale  salmon  color  when  cold.  Now  add  5  c.c.  of  a  freshly  prepared 
hot  aqueous  20%  solution  of  chemically  pure  lactose.  If  only  occasionally  using 
such  media,  tube  in  20  c.c.  quantities  and  add  one  drop  of  the  basic  fuchsin  and  four 
drops  of  the  sodium  sulphite  solution  and  i  c.c.  of  the  hot  freshly  prepared  lactose 
solution  to  a  tube  of  the  melted  agar  base  just  before  pouring  the  plate.  This  medium 
contains  i%  of  lactose.  Kendall  prepares  an  Endo  medium  which  only  contains 
i  1/2%  of  agar  and  with  a  reaction  just  alkaline  to  litmus  (about  plus  1.2%). 


28  CULTURE    MEDIA 

Colon  bacilli  show  on  this  medium  as  vermilion  colonies,  which  in  about  forty- 
eight  hours  have  a  metallic  scum  on  them.  Typhoid  and  dysentery  colonies  are 
grayish.  Streptococci  a  deep  red. 

FOR  LACTOSE  LITMUS  AGAR. 

Color  the  agar  base  with  litmus  solution  to  a  lilac  color.  Then  add  5  c.c.  of  the 
hot  freshly  prepared  lactose  solution  in  distilled  water.  This  may  be  tubed,  putting 
10  c.c.  in  each  test-tube,  or  put  in  quantities  of  50  or  100  c.c.  in  small  Erlenmeyer 
flasks.  It  is  then  sterilized  in  the  autoclave  (10  pounds  for  fifteen  minutes)  or  in 
the  Arnold. 

FOR  CONRADI-DRIGALSKI  MEDIUM. 

To  100  c.c.  of  lactose  litmus  agar  add  i  c.c.  of  a  solution  of  crystal  violet  (crystal 
violet  o.i  gram,  distilled  water  100  c.c.).  The  medium  is  then  ready  to  put  into 
plates.  Colon  colonies  are  pink.  Typhoid  and  dysentery  colonies,  a  bluish-gray. 

CONRADI'S  BRILLIANT  GREEN  MEDIUM. 

Take  of  Liebig's  extract  20  grams  (2%),  peptone  10  grams  (i%),  agar  30  grams 
(3%)and  water  to  1000  c.c.  This  amount  of  meat  extract  should  give  about  the 
proper  acidity,  +3.  If  not,  the  reaction  should  be  adjusted  to  that  point.  Filter 
through  cotton,  tube  150  c.c.  amounts  into  250  c.c.  Erlenmeyer  flasks  and  sterilize. 

Then  add  i  c.c.  of  a  i  to  1000  aqueous  solution  of  brilliant  green  (Hochst)  and 
i  c.c.  of  a  i%  solution  of  picric  acid  to  the  flasks  containing  150  c.c.  of  the  melted 
agar.  Sterilization  after  adding  the  dyes  precipitates  them  and  is  unnecessary. 
Pour  the  finished  medium  into  large  Petri  dishes  and  inoculate  the  surface  with  the 
faeces. 

Brilliant  green  does  not  interfere  with  agglutination  as  does  malachite  green. 

This  medium  is  considered  by  some  authorities  the  one  of  choice  in  isolating 
typhoid  bacilli  from  faeces  and  urine. 

The  surface  of  the  poured  plates  of  Endo,  Conradi-Drigalski,  and  the  brilliant 
green  media  should  be  dried  in  the  incubator  before  smearing  with  the  faeces.  For 
routine  work  I  prefer  Endo's  medium  followed  by  Russell's  double  sugar  agar. 

SELECTIVE  MEDIA  FOR  CHOLERA. 

Dieudonne's  medium  rests  on  the  ability  of  cholera  to  grow  when  alkali  is  present 
in  such  amounts  as  to  inhibit  the  growth  of  other  faecal  bacteria. 

Take  equal  parts  of  defibrinated  blood  obtained  at  the  slaughter  house  and 
normal  NaOH  solution.  Mix  30  parts  of  this  alkaline  blood  mixture  with  70  parts 
of  hot  3%  nutrient  agar.  The  poured  plates  should  be  left  half  open  over  night  in 
the  incubator  otherwise  even  cholera  will  not  grow  on  the  plates. 

Krumwiede  has  as  a  formula  for  his  medium  equal  parts  of  whole  egg  and  water, 
to  which  50%  water  egg  mixture  is  added  an  equal  amount  of  12  1/2%  crystal 
sodium  carbonate  solution.  This  alkaline  egg  mixture  is  steamed  for  20  minutes. 


MEDIA   FOR    PROTOZOA  2Q 

To  prepare  add  30  parts  of  this  alkaline  egg  mixture  to  70  parts  of  meat  extract  free 
3%  agar.  (No  meat  extract;  only  peptone  and  salt.)  The  cholera  colony  has  a 
hazy  look,  like  a  little  wad  of  absorbent  cotton  sticking  to  the  surface  with  a  metallic 
luster  halo. 

RUSSELL'S  DOUBLE  SUGAR  AGAR. 

A  fairly  stiff  agar  (2  to  3%)  with  a  reaction  of  about  plus  0.7  is  colored  with  litmus 
solution  to  produce  a  distinct  purple  violet  color.  It  may  be  necessary  to  add  more 
alkali.  To  this  litmus  tinted  agar  is  added  i%  of  lactose  and  0.1%  of  glucose  and 
the  medium  as  thus  prepared  is  tubed  and  slanted.  Sterilization  should  be  carried 
on  in  the  Arnold,  on  two  successive  days,  as  the  autoclave  temperatures  tend  to 
break  up  the  sugars. 

On  these  slants  typhoid  shows  a  delicate  growth  on  the  violet  slant  with  a  deep 
pink  in  the  butt  of  the  tube.  The  paratyphoids  show  gas  bubbles  in  a  pink  butt  with 
a  violet  slant. 

The  colon  bacillus  turns  both  slant  and  butt  a  deep  pink  and  the  butt  is  filled 
with  gas  bubbles.  To  inoculate  this  medium  we  take  material  from  a  suspicious 
colony  grown  on  Endo  and  smear  the  material  on  the  slant;  then  with  the  same 
platinum  needle  we  stab  into  the  butt. 

Culture  Media  for  Protozoa. 

MEDIUM  OF  MUSGRAVE  AND  CLEGG. 

Dissolve  in  1000  c.c.  of  water  0.3  to  0.5  gram  Liebig's  extract  and  0.3  to  0.5 
gram  of  common  salt.  If  desired  for  plating  add  2  to  3  %  of  agar. 

A  very  satisfactory  substitute  is  ordinary  nutrient  bouillon  diluted  one  to  ten. 

MEDIUM  OF  SMITH. 

Glucose  i.o  gram;  Peptone  i.o  gram;  NaCl  0.2;  Aqua  destill.  1000.0;  Na2CO3  0.3. 
Agar  q.  s.  is  added  for  solid  medium. 

MEDIUM  OF  CASTELLANI. 

This  is  an  aqueous  medium  containing  i%  of  lactose  and  10%  of  agg  albumin. 
This  may  replace  water  of  condensation  in  an  agar  slant. 

NOVY  MACNEAL  MEDIUM. 

Cover  125  grams  of  chopped  up  beef  with  1000  c.c.  of  water  and  place  over 
night  in  the  refrigerator.  Strain  and  add  20  grams  of  peptone,  5  grams  salt,  10  c.c. 
of  normal  sodium  carbonate  solution  and  20  to  25  grams  agar.  Prepare  as  for 
nutrient  agar  and  sterilize.  To  i  part  of  this  one-quarter  strength  meat  infusion 
nutrient  agar,  when  melted  and  cooled  down  to  60°  C.,  add  twice  its  volume  of  de- 


30  CULTURE    MEDIA 

fibrinated  rabbit's  blood.  This  medium  is  the  standard  one  for  the  culture  of  cer- 
tain trypanosomes  and  other  protozoa.  Under  the  designation  N.N.N.  medium 
(Nicolle  Novy  MacNeal)  Nicolle  has  modified  the  medium  so  that  there  is  only  salt 
and  agar  in  the  base  to  which  the  blood  is  added  instead  of  one  containing  meat  ex- 
tract and  peptone.  It  is  the  Hb  which  seems  essential  in  the  culture  of  various 
protozoa.  Rogers  used  citrated  salt  solution,  which  was  slightly  acidified  with  citric 
acid,  in  his  culturing  of  Leishmania  from  the  splenic  blood  of  cases  of  kala  azar. 
Incubation  at  22°  C. 

ROW'S    H^MOGLOBINIZED    SALINE    MEDIUM. 

Take  10  c.c.  blood  from  rabbit's  heart  or  arm  vein  of  man,  defibrinate  the  blood 
and  then  add  10  volumes  of  distilled  water  to  lake  the  cells  (liberation  of  Hb).  One 
volume  of  this  laked  blood  solution  is  added  to  two  volumes  of  sterile  1.2%  salt 
solution. 

CULTURE  MEDIA  FOR  TREPONEMATA. 

I.  NOGUCHI  formerly  first  inoculated  material  containing  treponemata  into  the 
testicle  of  rabbits,  obtaining  by  this  procedure  a  pure  culture,  after  a  few  transfers 
to  the  testicles  of  other  rabbits.     He  now  grows  the  organism  directly  from  serum 
from  a  chancre.     Test-tubes  2  by  20  cm.  are  filled  with  15  c.c.  of  a  medium  consist- 
ing of  2  parts  of  2%  slightly  alkaline  agar  to  which  when  melted  and  cooled  down 
to  50°  C.  is  added  i  part  of  ascitic  or  hydrocele  fluid.     At  the  bottom  of  the  medium 
in  the  tube  is  placed  a  fragment  of  fresh  sterile  tissue,  preferably  a  piece  of  rabbit's 
kidney  or  testicle.     After  the  medium  solidifies  a  layer  of  sterile  paraffin  oil  is  run 
in  so  that  it  covers  the  solid  medium  to  a  depth  of  3  cm.     Tne  material  is  inoculated 
at  the  bottom  of  the  tube  with  a  capillary  pipette.     Incubation  at  37°  C.  is  carried 
on  for  two  weeks.     The  tissue  acts  by  removing  any  oxygen  that  may  be  present  in 
the  depths  of  the  medium.    Anaerobiosis  is  a  necessary  condition.    Many  specimens 
of  ascitic  fluid  are  unsuited. 

II.  Serum  Agar  of  Muhlens  and  Hofmaim.— Fill  sterile  test-tubes  one-third 
full  with  horse  serum.     This  is  sterilized  on  three  successive  days  at  55°  C.     Then 
add  an  equal  amount  of  a  3%  agar  containing  0.5%  glucose  which  has  been  melted 
down  and  cooled  to  50°  C.     The  mixed  serum  agar  is  then  kept  at  55°  C.  for  two  hours. 
Such  tubes  are  inoculated  as  for  ascitic  agar  rabbit  tissue  media  and  incubated  under 
anaerobic  conditions,  preferably  in  a  flask  from  which  the  air  has  been  exhausted  and 
the  remaining  oxygen  absorbed  as  shown  in  the  anaerobic  bottle  described  and 
illustrated  in  Fig.  7. 

WELLMAN'S  PLACENTAL  AGAR. 

Fresh  human  placenta  is  thoroughly  ground  up  in  a  meat  chopper,  after  first 
washing  out  the  blood  by  running  sterile  salt  solution  through  the  attached  vessels. 
To  each  kilo  of  the  macerated  placental  tissue  is  added  i  liter  of  distilled  water. 
This  mixture  is  allowed  to  infuse  for  forty-eight  hours  at  refrigerator  temperature, 
after  which  it  is  passed  through  a  No.  N  Berkefeld  which  has  been  previously  tested 


PLACENTAL  AGAR  31 

and  found  to  hold  back  ordinary  bacteria.  The  first  half-hour's  nitrate  is  usually 
found  to  be  perfectly  sterile.  To  facilitate  this  filtration  the  cylinder  of  the  filter 
is  filled  with  fine,  clean,  sterile  sand  until  the  candle  is  completely  covered.  The 
filtrate  is  either  tubed  or  added  to  2%  sterile,  previously  melted  agar  at  40°  to  41°  C., 
mixed,  and  slanted.  No  titration  or  other  preparation  is  necessary,  except  that 
the  medium  is  placed  at  a  temperature  of  40°  C.  for  two  days  to  inactivate  the  comple- 
ment, as  suggested  by  Bass  in  the  use  of  human  blood  cultures.  Fresh  human 
placenta  contains  over  30%  of  the  hydrolytic  products  of  protein  digestion,  and  will 
therefore  secure  growths  of  strictly  parasitic  or  feebly  vegetative  bacteria,  and  possi- 
bly protozoa,  that  are  grown  with  great  difficulty  or  not  at  all  on  ordinary  media. 
For  instance,  the  acid-fast  organisms  from  bits  of  leprous  tissue,  either  of  human 
or  rat  origin,  grow  on  this  medium  so  readily  that  microscopic  growth  can  be  dis- 
cerned in  from  five  to  seven  days.  From  human  tuberculous  glands,  urine,  or  cerebro- 
spinal  fluid  the  same  method  will  give  a  growth  of  B.  tuberculosis  that  can  be  distin- 
guished in  from  seventy-two  hours  to  a  few  days. 


CHAPTER  III. 
STAINING  METHODS. 

IN  order  to  study  a  bacterial  or  blood  specimen  the  first  essential  is 
a  properly  prepared  film;  the  matter  of  staining  is  of  less  importance. 
The  slide  or  cover-glass,  after  cleaning  with  soap  and  water  or  by  special 
solutions,  should  be  polished  with  a  piece  of  old  linen.  If  a  glass  sur- 
face is  free  of  grease  a  loopful  of  water  will  smear  out  evenly  and  over 
the  entire  surface.  The  only  quick  practical  way  to  make  the  slide 
or  cover-glass  grease  free  is  to  burn  the  surface  for  a  moment  in  a  Bunsen 
or  alcohol  flame.  The  cover-glass  must  not  be  warped.  To  make  a 
preparation,  apply  a  small  loopful  of  distilled  water  on  the  slide  or  cover- 
glass  and,  touching  a  colony  with  a  platinum  needle,  stir  the  transferred 
culture  into  the  loopful  (not  drop)  of  water.  The  mistake  is  almost 
invariably  made  of  taking  up  too  much  bacterial  growth.  Fluid  cul- 
tures do  not  need  dilution.  Smearing  the  mixture  over  a  large  part  of 
the  cover-glass  or  over  an  equal  area  of  a  slide,  it  is  allowed  to  dry.  If 
very  little  water  is  used,  the  preparation  dries  readily.  Otherwise  it  can 
be  dried  in  the  fingers  high  over  a  flame.  As  soon  as  dry,  the  cover-glass 
should  be  passed  three  times  through  the  flame,  film  side  up,  to  fix  the 
preparation.  Slides  may  be  fixed  by  passing  them  five  times  through 
the  flame,  but  the  method  by  burning  alcohol  recommended  for  fixing 
blood-films  gives  more  satisfactory  bacterial  fixation.  For  routine  work 
the  stain  recommended  is  a  dilute  carbol  fuchsin.  Drop  about  5  to  10 
drops  of  water  on  the  cover-glass,  then  add  one  drop  of  carbol  fuchsin. 
Allow  the  dilute  stain  to  act  from  one  to  two  minutes,  then  wash  in 
water,  dry  between  small  squares  of  filter-paper  (4X4  inches),  and 
mount  in  balsam  or  the  oil  used  for  the  i/i 2-inch  immersion  objective. 

By  far  the  best  mounting  medium  is  liquid  petrolatum.  This  not  only  has  the 
advantage  of  always  being  of  proper  consistence  for  mounts,  as  opposed  to  Canada 
balsam,  which  must  frequently  be  made  thinner  with  xylol,  but  it  is  less  sticky  and 
does  not  develop  the  acidity  which  causes  balsam  mounts  of  Romanowsky  stains 
to  fade.  Furthermore,  it  has  superior  optical  qualities.  It  is  also  applicable  for 
mounting  small  insects  and  sporangia  of  moulds.  For  permanent  preparations  the 

32 


GRAM'S  STAINING  METHOD  33 

border  of  the  cover-glass  should  be  sealed  with  gold  size  or  some  other  cement. 
Some  prefer  to  mount  directly  in  water  without  preliminary  drying.  It  is  good 
practice  to  make  a  rule  to  always  keep  the  smeared  side  of  the  preparations  up — 
never  allowing  it  to  be  reversed.  By  this  simple  rule,  preparations  can  be  carried 
through  the  most  complicated  staining  methods  without  the  necessity  of  scratching 
the  cover-glass,  etc.,  to  see  which  is  the  film  side.  In  grasping  a  cover-glass  with  a 
Cornet  or  Stewart  forceps,  be  sure  that  the  tips  are  well  by  the  margin  of  the  glass, 
otherwise  the  stain  will  drain  off.  In  staining  with  slides,  the  grease  pencil  and  the 
glass  tubing,  as  recommednded  under  Blood  Smears,  will  be  found  useful.  The  dilute 
carbol  fuchsin  and  Lb'ffler's  methylene  blue  are  probably  the  best  routine  stains.  As 
a  rule  better  preparations  are  obtained  with  dilute  stains  than  with  more  concen- 
trated ones. 

Loffler's  Alkaline  Methylene  Blue. — Saturated  alcoholic  solution 
of  methylene  blue,  30  c.c. ;  one  to  ten  thousand  caustic  potash  solution, 
100  c.c.  (Two  drops  of  a  10%  solution  KOH  in  100  c.c.  of  water 
makes  a  i  :  10,000  solution.) 

Carbol  Fuchsin  (Ziehl-Neelsen). — Saturated  alcoholic  solution 
basic  fuchsin,  10  c.c.;  5%  aqueous  solution  carbolic  acid,  100  c.c. 

Gram's  Method. — The  most  important  staining  method  in  bac- 
teriological technic  and  the  one  so  rarely  giving  satisfactory  results  to 
the  inexperienced  is  Gram's  stain.  In  using  this  method,  the  following 
points  must  be  kept  in  mind: 

1.  Laboratory  cultures  (subcultures)  which  have  been  carried  over  for  years 
frequently  lose  their  Gram  characteristics. 

2.  Cultures  which  are  several  days  old  or  dead  or  degenerated  do  not  stain 
characteristically. 

3.  The  aniline  gentian  violet  deteriorates  when  exposed  to  light  in  two  or  three 
days — it  should  be  kept  in   the  dark.     It  should  have  a  rich,  creamy,   violet 
appearance. 

4.  The  iodine  solution  deteriorates  and  becomes  light  in  color.     It  should  be  of 
a  rich  port- wine  color. 

5.  The  decolorizing  with  95%  alcohol  should  stop  as  soon  as  no  more  violet 
stain  streams  out.     This  is  best  observed  over  a  white  background,  washing  at 
intervals.     Do  not  confuse  stain  on  forceps  for  that  on  preparation. 

6.  The  preparation  should  be  thin  and  evenly  spread.     Some  prefer  carbol 
gentian  violet   to   aniline  gentian  violet.     (Saturated   alcoholic  solution  of  gen- 
tian violet,  i  part;  5%  aqueous  solution  of  carbolic  acid,  10  parts.)     This  tends  to 
overstain. 

The  formula  for  aniline  gentian  violet  is  i  part  of  saturated  alcoholic  solution 
gentian  violet  and  3  parts  of  aniline  oil  water  (made  by  adding  2  c.c.  aniline  oil  to 
100  c.c.  distilled  water,  shaking  violently  for  three  to  five  minutes  and  then  filtering 
several  times  to  get  rid  of  the  objectionable  oil  droplets  which,  in  a  Gram-stained 
preparation,  show  as  confusing  black  dots). 
3 


34  STAINING   METHODS 

The  following  stock  solutions  of  Weigert  are  recommended: 

No.  i.  No.  2. 

Gentian  violet,  2  grams.              Gentian  violet,                2  grams. 

Aniline  oil,  9  c.c.                   Distilled  water,           100  c.c. 

Alcohol  (95%),  33  c.c. 

These  stock  solutions  keep  indefinitely.  Mix  i  c.c.  of  No.  i  with  9  c.c.  of  No.  2. 
Filter.  This  keeps  about  two  weeks  and  is  the  solution  to  pour  on  the  preparation. 
It  may  be  kept  on  from  two  to  five  minutes.  Some  hasten  the  staining  by  steaming 
as  for  tubercle  bacilli.  Next  wash  the  preparation  with  water  and  flood  the  cover- 
glass  with  Gram's  iodine  solution.  Some  bacteriologists  simply  pour  off  excess  of 
aniline  gentian  violet  and  immediately  drop  on  the  iodine  solution.  It  is  well  to 
repeat  the  application  of  the  iodine  solution  a  second  time.  The  iodine  solution  is  left 
on  one  minute  or  until  the  preparation  has  a  coffee-grounds  color. 

Gram's  Iodine  Solution. 
Iodine,  i  gram. 

Potassium  iodide,  2  grams. 

Distilled  water,  300  c.c. 

After  washing  off  the  excess  of  iodine  solution  at  the  tap,  drop  on  95%  alcohol 
and  decolorize  until  no  more  violet  color  streams  out.  Now  wash  again  and  counter- 
stain  either  with  the  dilute  Carbol  fuchsin  or  with  a  saturated  aqueous  solution  of 
Bismark  brown. 

The  Gram-positive  bacteria  are  stained  a  deep  violet. 

In  staining  smears  of  pus  for  gonococci  or  other  Gram-negative  bacteria  it  is 
best  to  first  stain  with  the  gentian-violet  solution  for  two  to  five  minutes.  Then 
wash  and  examine  the  preparation  mounted  in  water.  The  organisms  stand  out 
prominently.  After  noting  the  presence  of  the  cocci  treat  the  smear  with  the 
Gram  solution  and  proceed  as  in  the  usual  Gram  staining  technic. 

Stained  by  Gram's  Method.  Not  Stained  by  Gram's  Method. 

S.  pyogenes  aureus.  Meningococcus. 

S.  pyogenes  albus.  M.  catarrhalis. 

S.  pyogenes.  M.  melitensis. 

M.  tetragenus.  B.  typhosus. 

Pneumococcus.  B.  coli  communis. 

Anthrax  bacillus.  B.  dysenteriae  (Shiga). 

Tubercle  bacillus.  Sp.  cholerae  asiaticae. 

Lepra  bacillus.  B.  pyocyaneus. 

Tetanus  bacillus.  B.  mallei. 

Diphtheria  bacillus.  B.  pneumoniae  (Friedlander). 

B.  aerogenes  capsulatus.  B.  proteus. 

Oidium  albicans.  B.  of  influenza. 

Mycelium  of  actinomyces.  B.  of  bubonic  plague. 

Saccharomyces.  B.  of  chancroid. 

Hofman's  bacillus.  B.  of  Koch- Weeks. 

B.  xerosis.  Gonococcus. 


ACID-FAST   STAINING  35 

Practically  all  pathogenic  cocci  are  Gram-positive,  except  the  Gono- 
coccus,  the  Meningococcus,  the  M.  catarrhalis,  and  the  M.  melitensis. 

Practically  all  pathogenic  bacilli  are  Gram-negative,  except  the 
spore-bearing  ones  (exception  B.  malig.  cedemat.),  the  acid-fast  ones 
and  diphtheria  and  diphtheroid  organisms. 

The  bacillus  of  glanders  is  Gram-negative. 

Method  for  Staining  Acid -fast  Bacilli. — i.  Carbol  fuchsin,  with  gen- 
tle steaming  for  three  to  five  minutes  or  in  the  cold  for  fifteen  minutes. 

2.  Wash  in  water. 

3.  Decolorize  in  95%  alcohol  containing  3%  of  hydrochloric  acid 
(acid  alcohol),  until  only  a  suggestion  of  pink  remains — almost  white. 

4.  Wash  in  water. 

5.  Counterstain  in  saturated  aqueous  solution  of  methylene  blue 
or  with  Loffier's  methylene  blue. 

6.  Wash,  dry,  and  mount. 

The  steaming  of  the  slides  with  carbol  fuchsin  is  most  conveniently  carried  out 
by  resting  the  slides  on  a  piece  of  glass  tubing  bent  into  a  V  or  U  shape. 

A  method  in  which  the  organisms  or  granules  which  stain  by  the  Gram  method, 
and  to  which  so  much  importance  is  attributed  by  Much,  may  be  stained,  as  well 
as  those  retaining  acid-fast  properties,  has  been  proposed  by  Fontes.  The  method 
is  to  stain  the  preparation  with  carbol  fuchsin,  decolorize  with  acid  alcohol,  then  carry 
through  the  various  steps  of  the  Gram  method,  counters taining  however,  with  Bis- 
mark  brown.  Fontes  in  his  method  used  i  part  of  absolute  alcohol  and  2  parts  of 
acetic  acid  as  the  decolorizing  agent.  I  have  obtained,  however,  just  as  satisfactory 
results  with  the  acid  alcohol.  By  this  method  the  acid-fast  tubercle  bacilli  show  as 
red  rods  dotted  with  violet  granules.  Those  which  do  not  fully  retain  acid-fast 
properties  show  as  zigzag  violet  lines. 

Herman's  Stain  for  Tubercle  Bacilli. — It  has  been  claimed  that  this  stain  gives 
better  satisfaction  than  the  Ziehl-Neelsen.  It  consists*of  two  solutions:  (i)  ammo- 
nium carbonate  in  distilled  water,  i%;  (2)  crystal  violet  (methyl  violet  6B)  in  95% 
ethyl  alcohol,  3%.  The  two  solutions  are  kept  in  separate  bottles  and,  for  staining, 
i  part  of  (2)  is  mixed  with  3  parts  of  (i).  The  sections  are  placed  on  a  cover-glass, 
the  water  evaporated,  and  about  seven  drops  of  the  staining  mixture  are  placed 
on  the  specimen  and  allowed  to  steam  for  one  minute  over  a  water-bath.  Place  for 
a  few  seconds  in  10%  nitric  acid  and  then  in  95%  alcohol  to  decolorize.  Mount 
without  a  counterstain  or  use  eosin  i%  or  a  very  dilute  fuchsin.  The  organisms 
are  purple.  This  staining  method  may  be  applied  to  smears  of  concentrated  or 
unconcentrated  sputum  in  the  same  manner  as  for  sections  of  tissue. 

Smith's  formol  fuchsin : 

Saturated  alcoholic  solution  basic  fuchsin,  10  c.c. 

Methyl  alcohol,  10  c.c. 

Formalin,  10  c.c. 

Distilled  water  to  make  100  c.c. 


36  STAINING   METHODS 

This  gives  a  very  sharp  differentiation  of  bacteria  and  nuclear  structures.  It 
has  a  purplish  tinge.  Fixation  by  heat  gives  the  best  staining.  Allow  the  stain  to 
act  for  two  to  ten  minutes.  It  should  not  be  used  until  after  standing  twenty-four 
hours,  and  after  standing  about  two  weeks  it  appears  to  lose  its  sharp  staining  power. 

Archibald's  Stain. — This  is  an  excellent  bacterial  stain  and  has 
been  highly  recommended  by  Blue  and  McCoy  in  plague  work. 

Solution  No.  i.  Solution  No.  2. 

Thionin,  0.5  Methylene  blue,              0.5 

Phenol  crys.,  2.5  Phenol  cry s.,                    2.5 

Formalin,  i.o  Formalin,                         i.o 

Water,  100.0  Water,                          100.0 

Dissolve  for  twenty-four  hours.  Mix  equal  parts  and  filter.  Stain  smears 
fixed  by  heat  or  otherwise  for  ten  seconds. 

Nicolle's  Carbol  Thionin. 

Sat.  sol.  thionin  in  50%  alcohol,  10  c.c. 

Carbolic  acid  solution  (2%),  100  c.c. 

Pappenheim's  Stain. — Take  a  very  small  portion  of  methylene  green  on  the  point 
of  a  penknife  and  shake  it  into  a  test-tube.  Then  take  up  twice  as  much  pyronin 
and  deposit  it  in  the  same  test-tube.  Fill  the  test-tube  one-half  full  with  water  and 
the  solution  should  have  a  distinct  reddish-violet  color.  A  drop  on  a  piece  of  filter 
paper  shows  a  violet  center  and  peripheral  green  ring.  The  solution  should  be  fresh. 
Stain  from  two  to  five  minuted.  Differentiate  with  a  little  resorcin  on  a  penknife 
point  dissolved  in  one-quarter  of  a  test-tube  full  of  alcohol.  Dehydrate,  clear 
and  mount.  Polymorphonuclear  nuclei  stain  greenish;  nuclei  of  mononuclears  and 
plasma  cells  from  bluish-red  to  dull  violet.  Cytoplasm  of  lymphocytes  and  plasma 
cells  purplish-red.  Bacteria  red. 

Romanowsky  Stains. — See  under  section  on  Blood.  For  mount- 
ing specimens  showing  chromatin  staining,  as  malarial  parasites,  try 
panosomes,  intestinal  flagellates  etc.,  liquid  petrolatum  is  to  be  highly 
recommended.  The  chromatin  staining  lasts  without  any  fading  for 
at  least  two  years.  The  acidity  of  balsam  causes  rapid  fading  of  the 
chromatin. 

Neisser's  Stain  for  Diphtheria  Bacilli. 

Solution  No.  i.  Solution  No.  2. 

Methylene  blue,  o.igram.  Bismark  brown,  0.2 

Alcohol,  2  c.c.  Water  (boiling),  100  c.c. 

Glacial  acetic  acid,  5  c.c.  Dissolve  the  stain  in  the  boiling 

Distilled  water,  95  c.c.  water  and  filter. 

Dissolve  the  methylene  blue  in  the 
alcohol  and  add  it  to  the  acetic  acid 
water  mixture.     Filter. 


CAPSULE    STAINING  37 

To  stain:  Fix  the  preparation.  Pour  on  the  dilute  acetic  acid  methylene  blue 
solution  and  allow  to  act  from  thirty  to  sixty  seconds.  Wash.  Then  pour  on  the 
Bismark-brown  solution,  and  after  thirty  seconds  wash  off  with  water.  Dry  and 
mount.  The  bodies  of  the  bacilli  are  brown  with  dark  blue  dots  at  either  end. 

Neisser  recommends  only  five  seconds  as  the  time  of  application  of  each  solution. 
He  also  recommends  that  the  culture  be  only  nine  to  eighteen  hours  old  and  that  the 
temperature  of  the  incubator  shall  not  exceed  36°  C.  Incubation  at  37°  C.  gives 
satisfactory  results. 

Ponder's  Stain  For  Diphtheria  Bacilli. 

Toluidin  blue  (Grubler),  0.02  gram. 

Glacial  acetic  acid,  i  c.c. 

Absolute  alcohol,  2  c.c. 

Distilled  water  to  100  c.c. 

The  film  is  made  on  a  cover-glass  and  fixed  in  the  usual  way.  A  small  quantity 
of  the  stain  is  spread  on  the  film  and  the  cover-glass  is  turned  over  and  mounted  as 
a  hanging- drop  preparation.  The  metachromatic  granules  of  the  diphtheria  bacilli 
stain  with  striking  intensity.  With  diphtheroids,  the  more  intense  staining  sharply 
differentiates  from  ordinary  cocci  and  bacilli,  which  show  in  the  preparation  only 
as  faint  light  blue  bodies.  It  is  a  most  excellent  stain  for  bringing  out  the  ascopores 
of  yeasts.  In  my  opinion  the  stain  is  more  valuable  than  the  Neisser  method. 

Capsule  Staining. — The  best  method  for  studying  bacteria,  as  to 
presence  of  capsules,  is  in  the  hanging  drop,  with  the  greater  part  of  the 
light  shut  off  by  the  diaphragm. 

In  material  where  capsules  are  well  developed,  as  in  pneumonic  sputum,  the 
Gram  method  of  staining  brings  out  the  capsule  perfectly.  This  is  of  diagnostic 
value,  as  the  more  or  less  nonpathogenic  pneumococci  common  about  the  mouth 
do  not  seem  to  show  a  capsule  when  stained  in  this  way.  The  India  ink  method 
of  staining  gives  good  resalts  for  capsules. 

The  most  beautiful  method  of  staining  capsules  is  the  latest  one 
proposed  by  Muir. 

1.  Prepare  thin  film,  dry  and  stain  in  carbol  fuchsi  none-half  minute;  the  prepa- 
ration being  gently  heated  (steamed). 

2.  Wash  slightly  in  95%  alcohol,  then  wash  well  afterward  in  water. 

3.  Flood  preparation  in  mordant  for  five  to  ten  seconds. 

Mordant. — Sat.  aqueous  sol.  mercuric  chloride,  2  parts 

Tannic  acid  (20%  aqueous  sol.),  .2  parts 

Sat.  aqueous  sol.  potash  alum,  5  parts 

4.  Wash  in  water  thoroughly. 

5.  Treat  with  95%  alcohol  for  one  minute.     (The  preparation  should  have  a  pale 
red  color.) 

6.  Wash  well  in  water. 


38  STAINING   METHODS 

7.  Counterstain  with  methylene  blue  one-half  minute. 

8.  Dehydrate  in  alcohol.     Clear  in  xylol  and  mount.     (May  simply  dry  speci- 
men with  filter-paper.) 

Rosenow's  Capsule  Stain. — Make  a  very  thin  smear  of  the  pathological  material 
and  when  nearly  dry  cover  the  preparation  for  ten  to  twenty  seconds  with  10% 
tannic  acid  solution.  Wash  in  water  and  blot.  Stain  with  aniline  gentian  violet 
by  gently  steaming  for  one-half  to  one  minute.  Wash  in  water.  Apply  Gram's 
iodine  solution  for  one-half  to  one  minute.  Decolorize  in  95%  alcohol  and  then  stain 
with  alcoholic  solution  of  eosin.  Wash  in  water,  dry  and  mount. 

Flagella  Staining. — Inoculate  a  tube  of  sterile  water  (gently)  in 
upper  part,  with  just  enough  of  an  eighteen  to  twenty-four-hour-old 
agar  culture  to  produce  faint  turbidity.  Incubate  for  two  hours  at  37° 
C.  From  the  upper  part  of  culture  take  a  loopful  and  deposit  it  on  a 
cover-glass.  Dry  in  thermostat  for  one  to  five  hours  or  over  night. 
Use  perfectly  clean  cover-glasses.  To  stain  by 

Muir's  Modified  Pitfield  Method. 

i.  Flood  specimen  with  mordant.     Steam  gently  one  minute. 

Mordant. — Tannic  acid  (10%  aqueous  solution),  10  c.c. 

Sat.  aq.  sol.  mercuric  chloride,  5  c.c. 

Sat.  aq.  sol.  alum,  5  c.c. 

Carbol  fuchsin,  5  c.c. 

Allow  precipitate  to  settle  or  centrifuge.     Keeps  only  one  week. 

2.  Wash  well  in  water  for  two  minutes. 

3.  Dry  carefully — preferably  in  incubator. 

4.  Pour  on  stain.     Steam  gently  one  minute. 

Stain. — Sat.  aq.  sol.  alum,  10  c.c. 

Sal.  ale.  sol.  gentian  violet,  2  c.c. 

(May  use  carbol  fuchsin  instead  of  gentian  violet.) 
Stain  only  keeps  two  days. 

5.  Wash  well  in  water.     Dry  and  mount. 

Zettnow's  Flagella  Staining  Method. 

Solution  I. — Dissolve  2  grams  of  tartar  emetic  in  40  c.c.  water. 

Solution  II. — Dissolve  10  grams  tannin  in  200  c.c.  water.  To  the  200  c.c. 
solution  II,  warmed  to  50  or  60°  C.,  add  30  c.c.  of  the  tartar  emetic  solution. 
The  turbidity  of  the  mordant  should  entirely  clear  up  on  heating.  The  mordant 
should  keep  for  months  when  a  small  crystal  of  thymol  is  added  to  it. 

Next  dissolve  i  gram  silver  sulphate  in  250  c.c.  distilled  water.  Of  this  solution 
take  50  c.c.  and  add  to  it  drop  by  drop  ethylamine  (this  comes  in  a  33%  solution) 
until  the  yellowish-brown  precipitate  which  forms  at  first  is  entirely  dissolved  and 
the  fluid  is  entirely  clear.  It  requires  only  a  few  drops.  The  bacterial  preparations 
prepared  as  described  above  are  floated  in  a  little  mordant  contained  in  a  Petri  dish 


STAINING   OF  PROTOZOA  39 

which  is  heated  over  a  water  bath  for  five  to  seven  minutes.  Take  the  dish  contain- 
ing the  preparation  off  the  water  bath  and  as  soon  as  it  becomes  slightly  opalescent 
as  the  result  of  cooling  remove  the  cover-glass  preparation  and  wash  thoroughly  in 
water.  Then  heat  a  few  drops  of  the  ethylamine  silver  solution  upon  the  mordanted 
cover  preparation  until  it  just  steams  and  the  margin  appears  black.  Next  wash 
thoroughly  in  water  and  mount.  This  gives  the  most  satisfactory  results  of  any 
method  I  have  ever  experimented  with. 

Spore  Staining. — The  most  satisfactory  spore  staining  method  is 
really  the  negative  staining  of  the  spore  obtained  when  a  bac- 
terial preparation  is  stained  by  dilute  carbol  f uchsin  or  Loffler's  methy- 
lene  blue.  The  spore  appears  as  a  highly  refractile  piece  of  glass  in 
a  colored  frame. 

The  acid-fast  method,  as  for  tubercle  bacilli,  gives  good  results. 
The  decolorizing,  however,  must  be  lightly  done,  otherwise  the  spore 
will  lose  its  red  stain. 

Holler's  Method. — Fix  films  and  then  treat  with  chloroform  for  one  or  two 
minutes.  Wash  thoroughly  and  treat  with  a  5%  solution  chromic  acid  for  one 
minute.  Wash  in  water  and  then  stain  as  for  acid-fast  organisms  with  carbol 
f  uchsin.  Use  a  i%  sulphuric  acid  solution  instead  of  the  3%  acid  alcohol. 

Agar  Jelly  Staining  Method  of  H.  C.  Ross. 

Very  clear  i  1/2%  solution  of  agar  is  colored  with  Unna's  polychrome 
methylene  blue,  Giemsa's  solution,  thionin  or  Gram's  solution  of  iodine.  Very 
thin  smears  of  blood,  faeces  or  gastric  content  sediment  are  made  and  either 
fixed  lightly  in  the  flame  or  air  dried.  A  drop  of  the  melted  colored  agar  solu- 
tion is  placed  on  the  smeared  cover-glass  and  this  is  mounted  immediately  on  a 
clean  slide.  The  preparation  is  ready  for  examination  in  about  two  minutes. 

The  Staining  of  Protozoa. 

Unless  staining  albuminous  material  it  is  well  to  add  a  little  blood- 
serum  or  white  of  egg  to  the  preparation — about  one  loopful  to  a  smear. 
The  serum  or  white  of  egg  is  best  preserved  by  the  addition  of  2  %  chloro- 
form and  kept  tightly  corked. 

Giemsa's  Method. — Fix  moist  smears  with  a  fixative  made  by 
adding  i  part  of  95%  alcohol  to  2  parts  of  saturated  aqueous  solution 
of  bichloride  of  mercury.  Keep  in  this  solution  twelve  hours.  Now 
wash  for  a  few  seconds  in  water  and  then  for  about  five  minutes  with  a 
dilute  Lugol's  solution  (KI,  2  gm.;  Lugol's  solution,  3  c.c.;  Aqua,  100 
c.c.).  Now  wash  in  water  and  then  in  a  0.5%  solution  of  sodium  thio- 
sulphate  to  remove  the  iodine  which  was  used  to  remove  the  mercury. 


40  STAINING   METHODS 

Wash  in  water  five  minutes,  then  stain  with  Giemsa's  stain  as  used  in 
blood  work  for  one  to  ten  hours.  Wash  and  mount. 

Vital  Staining  of  Protozoa  with  Neutral  Red  Solution. — As  a  stock 
solution  one  uses  a  0.5%  aqueous  solution  of  neutral  red. 

The  drop  of  salt  solution  or  water  on  the  slide  should  be  tinged  a 
light  violet-rose  color  with  a  fraction  of  a  loopful  and  the  faeces  or  other 
material  emulsified  in  this. 

Protozoa  take  a  rose-pink  color  with  a  distinct  differentiation  be- 
tween endoplasm  and  ectoplasm. 

Should  the  faeces  be  quite  alkaline  the  neutral  red  will  be  decomposed 
with  the  formation  of  bilirubin-like  crystals. 

The  Giemsa  formalin  method  described  under  Blood  Work  is  of 
value  in  certain  cases. 

Highly  to  be  recommended  for  the  staining  of  protozoa,  whether  in  smears  or  in 
sections,  is  the  Panoptic  method. 

1.  Wright's  or  Leishman's  stain  for  one  minute. 

2.  Dilute  with  water  and  allow  dilute  stain  to  act  for  three  to  ten  minutes. 
Wash  in  water  and  then 

3.  Pour  on  dilute  Giemsa's  stain.     Allow  to  stain  from  thirty  minutes  to  twenty- 
four  hours.     Differentiate  with  i  :  1000  acetic  acid  solution  until  blue  stain  just 
shows  commencing  diffusion  into  the  acetic  acid.     Then  wash  in  water,  95% 
alcohol,  absolute  alcohol  and  treat  with  xylol  and  mount  in  liquid  petrolatum. 

With  preparations  other  than  blood  smears,  as  sections,  it  is  better  to  go  from 
95%  alcohol  to  oil  of  origanum,  then  mount. 

Owing  to  the  great  value  of  a  sharp  nuclear  picture  in  differentiating  amoebae 
it  is  of  great  importance  to  use  some  iron  haematoxylin  method.  That  of  Weigert 
is  given  in  the  appendix. 

Fix  moist  smears,  film  surface  down,  in  Zenker's  fluid  for  five  to  ten  minutes. 
Wash  in  water,  treat  with  Gram's  solution  and  wash  with  70%  alcohol  until  all  the 
yellow  color  is  discharged.  Wash  in  water.  Then  stain  with  Mallory's  phospho- 
tungstic  hamatoxylin  for  one-half  hour.  Wash  clear  and  mount.  See  appendix. 

Mallory's  Differential  Stain  for  Amoebae. — Staining  in  saturated  aqueous 
solution  thionin  for  from  three  to  five  minutes.  Next  differentiate  in  2%  aqueous 
solution  oxalic  acid  for  one-half  to  one  minute.  Then  wash  in  water,  clear  and  mount. 
Nuclei  of  amoebae  are  stained  a  brownish  red. 


CHAPTER  IV. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA— GENERAL 
CONSIDERATIONS. 

IN  order  to  study  bacteria  it  is  necessary  to  isolate  them  in  pure 
culture.  This  may  be  accomplished  by  taking  one  or  more  loopfuls  of 
the  material  and  mixing  it  in  a  tube  of  melted  agar  or  gelatin.  From 
this  first  tube  one  or  more  loopfuls  are  transferred  to  a  second  tube  of 
melted  agar  or  gelatin,  and  from  this  a  third  transfer  is  made,  thereby 
giving  us  tubes  in  which  the  distribution  of  the  bacteria  is  one  or  more 
hundred  times  less  in  the  second  than  in  the  first  tube,  and  equally 
more  dilute  in  the  third  than  in  the  second.  When  we  pour  the  con- 
tents of  the  tubes  into  Petri  dishes  we  would  have  the  bacterial  colonies 
on  the  first  plate  so  thick  that  it  would  be  impossible  to  pick  up  a  single 
colony  with  a  platinum  needle  without  touching  an  adjacent  one.  On 
the  second  plate  the  distribution  might  be  such  that  we  should  have 
discrete,  well  separated  colonies,  material  from  which  could  be  taken  up 
on  the  point  of  the  needle  or  loop  without  touching  any  other  colony. 
If  the  second  plate  did  not  meet  these  requirements,  the  third  would. 

In  clinical  bacteriology  we  work  almost  entirely  with  organisms 
preferring  blood-heat  temperature,  hence  it  is  necessary  to  use  agar 
or  blood-serum  as  standard  media  for  the  obtaining  of  isolated  colonies. 
Gelatin  is  of  little  value  for  this  purpose  in  medical  work.  In  using 
agar  it  will  be  remembered  that  it  solidifies  at  a  temperature  slightly 
below  40°  C.  and  does  not  melt  again  until  it  is  subjected  to  a  tempera- 
ture practically  that  of  boiling.  Again,  if  the  temperature  of  the  media 
exceeds  44°  C.  it  may  affect  injuriously  the  organisms  we  wish  to  study. 
Consequently  it  requires  careful  attention  and  quick  work  to  inoculate 
the  tubes,  mix,  transfer  and  pour  into  plates  within  the  limits  of  a  tem- 
perature which  injures  the  organisms,  and  one  which  brings  about  the 
solidification  of  the  agar. 

Again,  we  not  only  have  colonies  developing  from  organisms  which 
have  been  fixed  at  the  surface  as  the  agar  solidified  in  the  plate,  but 
more  numerous  ones  developing  from  bacteria  caught  in  the  depths 


42  STUDY   AND    IDENTIFICATION   OF  BACTERIA 

of  the  media.  Therefore  we  have  superficial  and  deep  colonies.  Ex- 
cept to  the  person  of  great  experience,  all  deep  colonies  look  alike  and 
there  is  at  times  great  difficulty  in  deciding  whether  a  colony  is  deep  or 
superficial.  It  is  in  the  matter  of  trying  to  obtain  information  from  the 
differences  in  deep  colonies  that  the  greatest  difficulties  in  the  study 


FIG.  8. — Petri  agar  plate.  Made  by  spreading  scrapings  from  the  mouth  over 
sterilized  nutrient  agar;  after  forty-eight  hours  in  the  thermostat  the  light  "colonies" 
develop.  Streaked  plate.  (Delafield  and  Prudden.} 

of  bacteriology  arise.  By  using  the  method  of  simply  stroking  plates 
along  five  or  six  parallel  lines  from  one  side  of  the  plate  to  the  other 
with  a  bent  glass  rod,  platinum  loop,  or  a  small  cotton  swab,  we  obtain 
colonies  which  are  well  separated  and  which  are  entirely  superficial. 
The  material  as  pus,  faeces,  throat  membrane,  etc.,  should  be  evenly 
distributed  in  a  tube  of  sterile  water  or  bouillon;  the  swab  which  was 


KOCH'S    SOLID    MEDIA  43 

originally  used  for  obtaining  the  material  being  then  pressed  against  the 
sides  of  the  test-tube  to  express  excess  of  fluid  and  then  stroked  gently 
over  successive  lines  on  one  plate.  Or,  if  the  organisms  be  very  abun- 
dant, over  a  second  plate  without  recharging  it  from  the  inoculated 
tube. 

According  to  my  experience  a  very  satisfactory  method  is  to  take  a  loopful  from 
the  bouillon  tube  suspension  of  the  pus  or  faeces  and  deposit  the  fluid  in  the  platinum 
loop  on  the  left  half  of  the  poured  plate  then,  without  recharging  the  locp.  we  touch 
the  right  half  of  the  plate.  Now  taking  a  bent  glass  rod  from  a  jar  of  95%  alcohol 
we  flame  it  and  to  cool  the  same  we  press  the  bent  portion  into  the  middle  of  the 
plate.  This  also  divides  the  surface  of  the  plate  into  two  portions.  Then  rubbing 
the  bent  rod  over  the  smaller  amount  of  the  material  on  the  right  side  we  carry  it 
over  the  entire  right  side.  Then  go  to  the  loopful  deposited  on  the  left  side  with  the 
rod  and  rub  it  over  this  side.  For  urine,  deposit  one  drop  on  one  side  and  5  drops 
on  the  other.  A  smear  from  pus,  sputum,  urine  or  throat  culture  should  always  be 
made  first  in  order  to  get  an  idea  as  to  the  degree  of  dilution  which  is  necessitated 
before  plating  out. 

To  obtain  isolated  colonies  on  blood-serum  or  blood-streaked  agar, 
which  can  be  touched  and  by  transfer  obtained  in  pure  culture,  we 
simply  smear  the  material  on  a  slant  of  either  medium.  Then,  without 
sterilizing  the  loop,  we  smear  it  thoroughly  over  a  second  slant,  and  so  on 
to  a  third,  or  possibly  a  fourth  or  fifth. 

At  present  the  classification  of  the  bacteria  is  very  unsatisfactory 
from  a  scientific  standpoint.  The  nomenclature  abounds  in  instances 
where  three  or  four  terms  are  used  in  naming  a  single  bacterium,  in- 
stead of  the  single  generic  name  and  single  specific  one  as  is  used  in 
zoological  nomenclature.  This  matter  of  nomenclature  is  a  subordi- 
nate factor  in  the  confusion  when  we  begin  to  investigate  and  find  that 
different  names  have  been  applied  to  apparently  the  same  organism. 
The  slightest  variation  in  morphological,  locomotor,  or  biological 
characteristics  seems  to  be  considered  sufficient  by  many  observers  to 
justify  the  description  of  a  new  species,  and,  of  course,  the  giving  of  a 
new  name.  Many  of  these  names  which  are  now  retained  were  applied 
prior  to  the  epoch-making  introduction  of  gelatin  media  by  Koch  (1881) 
and  consequently  at  a  time  when  the  isolation  of  organisms  in  pure 
culture  was  a  matter  of  extreme  difficulty  and  uncertainty.  One  of 
the  first  facts  noted  by  the  student  in  taking  up  bacteriology  is  the 
difficulty  in  determining  motility;  this  property  should  always  be 
tested  on  young  cultures  in  bouillon.  In  Brownian  movement  there 
is  a  sort  of  scintillating  movement,  but  the  bacterium  does  not  move 


44 


STUDY   AND   IDENTIFICATION   OF  BACTERIA 


from  that  part  of  the  field.  In  current  movement  all  the  bacteria 
swarm  in  the  same  direction,  going  very  fast  at  times,  and  then  more 
slowly.  If  in  great  doubt,  the  mounting  of  the  organisms  in  a  2% 
solution  of  carbolic  acid  will  stop  movement  if  it  be  true  functional 
motility,  while  Brownian  and  current  movement  are  not  interfered 
with.  In  true  motility  bacteria  move  in  opposite  and  in  all  directions, 


FIG.  9. — Chart  in  use  at  the  U.  S.  Naval  Medical  School. 

and  move  away  from  the  place  where  first  observed  unless  degenerated 
or  dead. 

At  times  we  judge  of  motility  by  the  presence  of  this  characteristic  in  a  few  of 
the  organisms  seen  in  the  microscopic  field,  the  vast  majority  of  the  bacteria  not 
showing  motility.  A  source  of  error  can  be  present  when  the  bacteria  are  emulsified 
in  a  drop  of  water  which  might  contain  motile  bacteria. 


CHARACTERISTICS    OF  BACTERIA  45 

Reaction  of  media  is  of  the  greatest  importance  in  causing  variation 
in  the  functions  of  bacteria,  and  is  one  which  has  until  recently  been 
almost  entirely  neglected.  In  describing  an  organism  at  the  present 
time  it  is  always  necessary  to  note  the  reaction  of  the  media,  the  tem- 
perature at  which  cultivation  took  place,  and  the  age  of  the  culture 
when  examined. 

In  the  following  keys  the  term  bacterium  has  been  used  as  a  general 
designation  for  all  schizomycetes.  Migula  calls  motile  rod-shaped 
organisms  bacilli,  and  nonmotile  ones  bacteria.  Lehmann  and  Neu- 
mann call  spore-bearing  organisms  bacilli,  and  nonspore-bearing  ones 
bacteria. 

The  B.  typhosus  is  very  motile  and  does  not  possess  spores.  Accord- 
ing to  Migula,  it  would  be  the  Bacillus  typhosus;  according  to  Lehmann 
and  Neumann,  the  Bacterium  typhosum.  The  B.  anthracis  has  spores 
and  is  nonmotile.  Hence  it  would  be  Bacterium  anthracis,  according 
to  Migula,  and  Bacillus  anthracis,  according  to  Lehmann  and  Neumann. 

In  the  use  of  the  keys  at  the  head  of  each  group  of  organisms  it  will 
be  observed  that  the  primary  separation  is  on  the  basis  of  morphology 
—the  cocci  in  one  group,  the  bacilli  in  three  subgroups:  one  for  those 
rod-shaped  organisms  showing  branching  and  curving  forms,  one  for 
the  spore  bearers  and  one  for  the  simple  rods.  The  spirilla  are  grouped 
by  themselves. 

An  important  method  of  differentiation  is  the  reaction  to  Gram's 
stain.  It  should  be  remembered  that  organisms  carried  along  on  arti- 
ficial media  often  lose  their  Gram  staining  characteristics;  hence  it  is 
desirable  to  determine  this  staining  reaction  in  cultures  freshly  isolated. 
Be  sure  that  the  stains,  especially  the  aniline  gentian  violet  and  the 
iodine  solution,  have  not  deteriorated.  There  is  no  more  important 
stain  than  this,  and  none  which  requires  greater  experience.  The  chief 
causes  of  conflicting  results  are  i.  working  with  old  cultures  and  2.  not 
having  satisfactory  staining  solutions. 

Motility,  as  stated  above,  is  at  times  difficult  to  determine.  For  this 
purpose  young  eighteen-hour-old  bouillion  cultures  are  preferable,  and 
the  preparation  should  be  made  by  applying  a  vaseline  ring  to  the  slide, 
then  putting  a  drop  of  the  bouillion  culture  in  the  center  of  the  ring 
(or  a  drop  of  water  inoculated  from  an  agar  slant  growth),  then  putting 
on  a  cover-glass.  By  this  method  current  movement  is  done  away  with 
and  the  preparation  keeps  for  hours.  This  is  a  convenient  method  for 
agglutination  tests. 


46  STUDY   AND   IDENTIFICATION   OF  BACTERIA 

Liquefaction  of  gelatin  is  a  very  important  means  of  differentiating.  When  a 
room-temperature  incubator  is  not  at  hand  (20°  to  22°  C.),  it  is  better  to  put  the 
inoculated  gelatin  tube  in  the  body-temperature  incubator,  and  from  day  to  day  test 
the  power  of  solidifying  with  ice-water.  If  the  organism  digests  the  gelatin  (a 
liquefier),  the  medium  will  remain  fluid  when  placed  in  ice- water;  if  the  organism 
is  a  nonliquefier,  the  medium  in  the  tube  becomes  solid.  Of  course  we  lose  the 
information  to  be  obtained  from  the  shape  of  the  area  of  liquefaction. 


FIG.  10. — Series  of  stab  cultures  in  gelatin,  showing  modes  of  growth  of  different 
species  of  bacteria.     (Abbott.) 

For  routine  work  the  only  sugar  media  used  are  the  glucose  and  the 
lactose  bouillon.  These  are  of  the  utmost  importance  in  differentiating 
organisms  of  the  typhoid  and  colon  group.  Following  Ford,  these 
intestinal  bacteria  have  primarily  been  separated  by  their  action  on 
litmus  milk — whether  turning  it  pink  or  only  slightly  changing  or  not 
changing  at  all  the  original  color. 

Examine  the  colonies  on  Petri  plate  at  first  with  the  unaided  eye, 
then  with  a  hand  magnifying  glass  or  low-power  objective,  using  re- 


KOCH'S   POSTULATES  47 

fleeted  and  transmitted  light  alternately.  Having  determined  the 
presence  of  two  or  more  different  kinds  of  colonies,  make  a  ring  with 
wax  pencil  around  one  or  more  of  each  kind  of  colony,  numbering  them. 
The  slides  or  culture  tubes  used  in  determining  the  species  of  organism 
present  in  the  plate  should  bear  the  same  number  as  that  of  the  colony 
from  which  the  material  was  taken.  A  convenient  procedure  is  to  put 
a  loopful  of  water  on  a  clean  cover-glass  and  to  emulsify  material  from 
a  colony  in  it.  Then  invert  over  a  concave  slide  without  vaselining 
the  circumference  of  the  concavity.  After  examining  for  motility, 
smear  out  and  dry  the  bacterial  preparation.  Then  fix  in  the  flame 
and  stain  with  aniline  gentian  violet  for  two  to  five  minutes.  Wash 
and  mount  the  preparation  in  water.  Afterward  pass  through  the  usual 
Gram  technic. 

After  this  inoculate  the  various  culture  media  from  similar  colonies. 
One  may  inoculate  a  tube  of  bouillon  from  a  single  colony  and  later 
on  inoculate  the  other  culture  tubes. 

In  testing  for  gas  production  it  is  better  to  use  the  Durham  fermenta- 
tion tube  as  small  amounts  of  gas  may  not  be  easily  detected  with  deep 
stab  cultures  into  glucose  or  lactose  agar. 

If  a  Durham  or  Smith  tube  be  not  at  hand  the  production  of  gas  may  be  deter- 
mined by  observing  bubble  formation  on  the  surface  of  the  sugar  bouillon  culture. 
As  none  of  the  pathogenic  cocci  produce  gas,  fermentation  tubes  are  unnecessary 
where  cocci  are  to  be  studied.  The  litmus  milk  tube  gives  data  as  to  acid  production. 

An  important  point  is  to  wait  at  least  forty-eight  hours  (in  the  case 
of  M.  melitensis,  four  to  seven  days)  before  reporting  on  the  cultural 
findings  on  the  agar  or  blood-serum  slant  or  plate  upon  which  the 
material  is  smeared  (pus,  exudate,  blood,  etc.). 

Should  an  organism  be  encountered  in  original  investigations  these 
requirements  as  to  etiological  relationship  should  be  carried  out  (Koch's 
postulates),  i.  The  organism  should  be  constantly  present  in  that 
particular  pathological  condition.  2.  Such  bacteria  should  be  isolated 
in  pure  culture  from  the  pathological  material.  3.  Such  pure  cultures 
when  inoculated  into  suitable  animals  should  reproduce  the  pathologi- 
cal conditions  and  should  be  capable  of  a  second  isolation  in  pure  culture 
from  such  an  experimental  animal.  For  various  reasons,  such  as  unsuit- 
able animals  or  artificial  media,  these  requirements  are  impossible  of 
execution  with  several  organisms  which  are  generally  recognized  as  the 
causes  of  certain  diseases. 

The  experimental  animals  most  frequently  employed  in  the  diagno- 


48  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

sis  of  bacterial  diseases  are  the  guinea  pig,  the  rabbit,  the  white  rat 
and  the  white  mouse.  In  the  following  diseases  the  most  suitable 
animals  for  inoculation  are : 

1.  Tetanus — mice  or  guinea-pigs  subcutaneously.     The  spasms  begin  in  the  limbs 
nearest  the  site  of  inoculation. 

2.  Pneumococci  and  streptococci — mice   intraperitoneally  or  rabbits  intraven- 
ously. 

3.  Staphylococci — rabbits. 

4.  Diphtheria,   tuberculosis,   anthrax  and    malignant    oedema — the  guinea-pig 
subcutaneously. 

5.  Glanders  and  cholera — the  guinea  pig,  intraperitoneally. 

6.  Plague — guinea-pigs,  cutaneously  or  subcutaneously. 

In  the  cutaneous  method  of  infection  the  material,  as  from  a  plague  bubo,  or 
the  sputum  from  pneumonic  plague,  is  thoroughly  rubbed  with  a  glass  rod  upon 
the  shaven  surface  of  the  guinea-pig. 

In  the  subcutaneous  method  one  can  use  a  hypodermic  needle  (the  all  glass 
syringe  with  platino-iridium  needle  is  the  best)  or  an  opening  can  be  cut  with  the 
scissors,  a  pocket  then  opened  up  with  the  forceps  and  a  piece  of  tissue  inserted  to 
the  bottom  of  the  pocket  with  the  forceps. 

The  large  ear  vein  of  the  rabbit  is  used  for  intravenous  inoculation.  This  can  be 
made  to  stand  out  with  either  hot  water  or  xylol. 

In  intraperitoneal  injections  the  animal  is  best  held  head  down  so  that  the 
intestines  gravitate  downward.  The  shaven  skin  is  pinched  up  and  the  needle 
inserted  in  the  median  line. 


CHAPTER  V. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA— COCCI.    KEY 

AND  NOTES. 

Streptococcus  Forms. — Cells,  divide  to  form  chains. 

I.  Gelatin  not  liquefied. 

1.  Haemolytic  zone  on  blood  agar. 

a.  Very  slight  acidity  in  lactose  litmus  bouillon.     S.  pyogenes.     Tends  to 
produce  arthritis  in  experimental  animals.     Often  a  granular  sediment  in 
bouillon. 

b.  Marked  acidity  but  no  gas  production  in  lactose  litmus  bouillon.     S. 
acidi  lactici.    Non  pathogenic.     Forms  diffuse  cloudiness  in  bouillon. 

2.  Greenish  appearance  about  colonies  on  blood  agar. 

a.  No  tendency  to  capsule  formation.     S.  viridans.  Produces  endocarditis 
in  experimental  animals. 

b.  Distinct  capsule  formation  in  pathological  material  or  on  favorable  media. 
S.  lanceolatus  (Pneumococcus).     Gram  positive,  lance-shaped  cocci  with 
bases  apposed  within  a  capsule. 

c.  Very  marked  capsule  development  on  all  media.     S.  mucosus.  A.  strepto- 
coccus with  extraordinary  capsule  development,  up  to  io(i  in  width,  S. 
mesenterioides,  is  not  pathogenic. 

II.  Gelatin  liquefied. 

Streptococcus  coli  gracilis.     (Cocci  quite  small — 0.2  to  0.4/1.     In  faeces.) 
A  tube-like  liquefaction,  chains  rather  long;    only  slight  growth  on  agar. 

Constant  inhabitant  of  stools  of  meat  diet. 
Sarcina  Forms. — Cells  divide  in  three  dimensions  of  space.     (Packets). 

A.  No  pigment  production  on  agar. 

a.  Sarcina  alba.     (Colonies  finely  granular.) 

b.  Sarcina  pulmonum. 

B.  Yellowish  pigment. 

a.  Sarcina  lutea.     (Colonies  coarsely  granular.) 

b.  Sarcina  flava.     (Colonies  finely  granular.) 

C.  Rose-red  pigment. 

a.  Sarcina  rosea. 

Micrococcus  Forms. — Cells  divide  irregularly  in  various  directions. 
I.  Gram-positive  cocci. 
A.  Cocci — round. 

i.  Divide  in  two  planes  at  right  angles.     Tetrad  formation.     Merismopedia. 
a.  M.    tetragenus.     Moist    white    viscid    colones.     No    liquefaction    of 
gelatin.     Capsule. 

4  49 


50  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

2.  Divide  irregularly.     Bunch  of  grapes  arrangement.     (Staphylococci.) 

a.  Gelatin  not  liquefied.     M.  cereus  albus. 

b.  Gelatin  liquefied.       /  M.  (Staphylococcus)  pyogenes  albus. 

\  M.  (Staphylococcus)  pyogenes  aureus. 

c.  Gelatin  very  slightly  liquefied. 

S.  epidermidis  albus.     (Stitch  coccus.) 
B.  Cocci — biscuit-shape. 

Diplococcus  crassus.     (May  be  mistaken  for  meningococcus.) 
On  ordinary   agar  we  have  a  scanty  growth  resembling  the  streptococcus. 
Colonies  on  ascites  agar  are  smaller  than  those  of  meningococcus.     It  produces  acid 
in  glucose,  maltose  and  lactose. 
II.  Gram-negative  cocci. 

A.  Grow  only  at  about  incubator  temperature. 

1.  Grow  only  on  blood  or  serum  media.     Gonococcus. 

2.  Grow  on  blood  serum  media,  or  glycerine  agar. 

a.  Diplococcus  intracellularis  meningitidis.     (Produces  acid  in  glucose 
and  maltose  but  not  in  lactose.) 

3.  Grows  on  ordinary  media.     Micrococcus  melitensis. 

B.  Will  grow  at  room  temperature  as  well  as  at  37°  C. 

a.  Micrococcus  catarrhalis.     Does  not  produce  acid  in  glucose  or  maltose. 

b.  M.  pharyngis  siccus.     Colonies  dry  and  tough  and  adhere  to  medium. 
NOTE. — Other  biscuit-shaped  Gram  negative  organisms  resembling  the  meningo- 
coccus are  (a)  Diplococcus  flavus.     The  colonies  show  yellow  pigment  and  we  have 
three  varieties  according  to  the  depth  of  the  yellow  color,     (b)  M.  pharyngis  siccus 
and  (c)  M.  cinereus  chiefly  have  coarse  dry  colonies  on  ascitic  agar. 


STREPTOCOCCUS  FORMS. 

Those  cocci  tending  to  arrange  themselves  in  chains  are  usually 
described  as  streptococci.  (Ogston,  1881;  Rosenbach,  1884.) 

When  we  consider  that  certain  bacilli  at  times  assume  an  arrange- 
ment which  we  term  strepto-bacilli,  yet  have  no  relationship,  it  would 
suggest  that  the  matter  of  chain  morphology  is  simply  a  characteristic 
common  to  many  entirely  different  cocci. 

Again  old  laboratory  cultures  of  streptococci  may  show  alternations  of  cocci 
and  rods  giving  the  appearance  of  the  dots  and  dashes  of  the  Morse  code.  Further- 
more unsuitable  media  may  bring  about  various  involution  types  in  an  organism 
primarily  streptococcal. 

It  is  often  difficult  to  distinguish  streptobacilli  from  streptococci  morphologic- 
ally and  the  same  is  true  of  diplococci  and  diplobacilli.  These  bacillary  pairs  and 
chains  however  often  show  bipolar  staining  and  are  almost  invariably  Gram 
negative. 

While  streptococci  tend  to  assume  chain  formation  in  pus  and  tissues  they  often 
appear  as  diplococci  in  blood. 


STREPTOCOCIC  51 

The  essential  point  to  bear  in  mind  is  that  the  finding  of  a  strepto- 
coccus does  not  necessarily  explain  an  infection,  because  normally 
streptococci  are  among  the  organisms  most  frequently  and  abundantly 
found  in  plates  made  from  normal  buccal  and  nasal  secretions.  It  is 
well  to  be  very  conservative  when  reporting  streptococci  as  the  etiolog- 
ical  factor  from  lesions  of  the  throat  or  nose. 

Probably  the  most  practical  point  in  the  differentiation  of  strepto- 
cocci, next  to  that  of  pathogenicity,  is  the  occurrence  of  long  or  short 
chains,  the  virulent  ones  tending  to  appear  in  chains  of  from  ten  to 
twenty  cocci,  while  the  normal  inhabitants  of  the  nose,  mouth  and 
faeces  generally  tend  to  be  in  shorter  chains. 


FIG.   ii — Streptococcus  pyogenes.     (Kolle  and  Wassermann.) 

As  regards  virulence,  this  is  exceedingly  variable — it  is  soon  lost, 
but  may  be  restored  either  by  inoculating  streptococci  along  with 
various  other  organisms  or  by  passage  through  successive  rabbits. 
The  rabbit  is  the  most  susceptible  animal  and  should  be  inoculated  in 
one  of  the  prominent  ear  veins.  If  the  needle  of  the  syringe  is  not  in- 
serted in  the  vein  it  will  be  difficult  to  force  in  the  material  and  a  swell- 
ing will  immediately  show  itself. 

Besides  the  morphological  and  pathogenic  variations,  Schottmuller 
has  noted  differences  where  these  organisms  are  grown  on  i  part  of 
blood  and  3  to  6  parts  of  agar.  On  this  medium  Strep,  erysipelatis 
has  a  hemolytic  action,  the  laking  of  the  red  cells  bringing  about  a  more 
or  less  clear  ring  surrounding  the  colony.  The  short-chain  strepto- 
cocci do  not  have  a  hemolytic  halo.  The  pneumococcus  has  a 


52  STUDY   AND    IDENTIFICATION    OF  BACTERIA 

greenish  zone.  Streptococci  which  are  profoundly  toxic  and  which 
have  been  isolated  from  milk-borne  epidemic  sore  throats  differ  from 
the  ordinary  S.  pyogenes  in  being  encapsulated,  not  tending  to  form 
chains  and  producing  only  slight  haemolysis  on  blood  agar. 

Some  of  the  English  authorities  have  introduced  biochemical 
methods  of  differentiating:  the  Strep,  pyogenes  coagulating  milk,  re- 
ducing neutral  red,  and  producing  acid  in  lactose,  saccharose,  and 
mannite  media. 

S.  pyogenes  does  not  produce  acid  in  inulin  media  while  the  pneu- 
mococcus  does, 

A  freshly  prepared  solution  of  sodium  taurocholate,  5%,  added  to  an  equal 
amount  of  a  twenty-four-hour  bouillon  culture  of  S.  pyogenes  does  not  disintegrate 
the  cocci  or,  at  any  rate,  not  within  a  few  minutes.  The  reverse  is  true  of 
the  pneumococcus. 

When  we  consider  the  biochemical  variations  which  a  single  organism,  as  the 
colon  bacillus,  may  exhibit,  the  value  of  such  methods  of  differentiating  may  well 
be  questioned.  The  question  of  the  symbiotic  relationship,  which,  when  established 
between  two  or  more  bacteria,  may  cause  harmless  organisms  to  take  on  virulence, 
would  appear  to  be  a  more  important  consideration. 

Almost  without  exception,  human  streptococci  are  Gram  positive. 
Their  colonies  are  quite  small,  but  distinct  and  discrete.  In  appear- 
ance the  colonies  of  streptococci  and  pneumococci  are  practically  iden- 
tical. In  a  blood-serum  throat  culture  pneumococcus  and  streptococcus 
colonies  are  the  smallest,  diphtheria  ones  are  quite  small  and  discrete, 
but  slightly  flatter.  (Always  examine  the  water  of  condensation  for 
streptococci.)  The  sarcina  and  staphylococcus  colonies  are  much 
larger. 

Streptococcic  colonies  on  blood  agar  are  much  more  moist  and  luxuriant  than 
on  ordinary  agar.  A  very  important  point,  in  judging  whether  a  streptococcus 
or  other  organism  is  pathogenic  in  a  given  infection,  is  to  examine  smears  from  the 
pus  or  other  material  in  a  Gram-stained  specimen  for  information  as  to  abundance 
and,  in  particular,  phagocytosis  of  any  organism,  before  plating  out. 

Streptococci  are  commonly  the  cause  of  diffuse  phlegmonous  inflam- 
mations, while  the  staphylococci  cause  circumscribed  lesions.  Strepto- 
cocci cause  necrosis  and  do  not  characteristically  produce  pus.  The 
importance  of  the  streptococcus  as  a  secondary  infection  in  diphtheria, 
tuberculosis,  small-pox,  and  even  in  typhoid  fever  must  always  be  kept 
in  mind.  It  is  this  infection  which  does  not  respond  to  diphtheria 
antitoxin,  and  not  the  diphtheria  one. 


SARCINA   FORMS  53 

When  freshly  isolated  from  human  lesions  streptococci  often  show  only  a  slight 
virulence  for  animals.  Hence  massive  doses  are  indicated  and  intravenous  or 
intraperitoneal  injections.  The  guinea-pig  is  not  very  susceptible  to  streptococci; 
the  rabbit  and  white  mouse  being  the  animals  of  choice. 

In  nondiphtheritic  anginas,  puerperal  fever,  ulcerative  endocarditis 
and  coccal  enteritis  it  is  the  streptococcus  which  is  usually  the  cause. 
It  has  been  claimed  that  acute  articular  rheumatism  is  due  to  a  short- 
chain  streptococcus  (M.  rheumaticus),  which  is  best  isolated  from 
material  from  an  acute  joint  infection,  but  may  also  be  isolated 
occasionally  from  the  blood.  It  produces  much  acid  and  clots  milk  in 
two  days.  The  growth  is  described  as  being  more  luxuriant  than  that 
of  S.  pyogenes.  It  is  about  0.5/4  in  diameter. 

The  majority  of  investigators  have  reported  streptococci  from  acute  joint  inflam- 
mations and  bacilli  from  chronic  infectious  joint  affections.  Goadby  has  considered 
a  streptobacillus,  somewhat  resembling  Ducrey's  bacillus  of  chancroid,  which 
exhibits  marked  pleomorphism  and  Gram  variations,  and  grows  best  on  egg  albumin 
agar  of  plus  3  reaction  as  the  cause  of  arthritis  deformans  and  alveolar  osteitis. 
Inoculation  of  cultures  of  this  organism  into  or  around  the  knee-joints  of  rabbits 
has  produced  lesions  similar  to  those  of  rheumatoid  arthritis. 

SARCINA  FORMS. 

These  are  best  observed  in  hanging-drop  preparations,  when  they 
can  be  seen  as  little  cubes,  like  a  parcel  tied  with  a  string,  and  by  noting 
them  when  turning  over,  it  will  be  seen  that  they  are  different  from  the 
tetrads  which  only  divide  in  two  directions  of  space.  At  times  the 
packet  formation  is  not  perfect  and  it  will  be  difficult  to  distinguish 
such  as  sarcinae.  All  sarcinae  stain  by  Gram.  If  the  staining  of 
sarcinae  be  too  deep  it  may  obscure  the  lines  of  cleavage.  Sarcinae  are 
nonmotile. 

Various  sarcinae  have  been  isolated  from  the  stomach,  especially 
when  there  is  stagnation  of  stomach  contents.  Sarcinae  have  also  been 
found  in  the  intestines.  In  plates  the  S.  lutea  is  frequently  a  contami- 
nating organism,  being  rather  constantly  present  in  the  air.  The 
demonstration  of  sarcina  morphology  should  always  be  made  from 
liquid  media,  as  bouillon.  Urine  makes  an  excellent  medium. 

MICROCOCCUS  FORMS. 

This  grouping  includes  all  cocci  which  do  not  show  chain  or  packet 
formation.  It  will  be  found  convenient  to  divide  them  into  two  classes 


54 


STUDY   AND    IDENTIFICATION   OF  BACTERIA 


according  to  their  staining  by  Gram.  The  M.  tetragenus,  S.  pyogenes 
aureus  and  the  pneumococcus  stain  by  Gram,  while  the  gonococcus, 
the  meningococcus,  the  M.  catarrhalis  and  the  M.  melitensis  are  Gram 
negative. 

M.  tetragenus. — This  organism  is  frequently  found  associated  with 
other  organisms  in  sputum,   especially  with  tubercle  and  influenza 
bacilli.     The  colonies  are  white,  slightly  smaller 
than  staphylococci  and  are  quite  viscid. 

It  was  formerly  considered  unimportant  in 
disease,  but  the  idea  now  prevails  that  it  is  re- 
sponsible for  many  abscesses  about  the  mouth, 
especially  in  connection  with  the  teeth.  Injected 
subcutaneously  into  mice,  it  produces  a  septicaemia 
and  death  in  three  or  four  days.  The  blood  shows 
great  numbers  of  encapsulated  tetrads.  It  has  been 
reported  twice  as  a  cause  of  septicaemia  in  man. 

Staphylococci. — To  cocci  dividing  irregularly 
and  usually  forming  masses  which  are  likened  to 
clusters  of  grapes  the  term  staphylococcus  is  ap- 
plied. While  there  have  been  experiments  which 
show  that  by  selecting  pale  portions  of  a  yellow 
colony,  eventually  a  white  colony  could  be  pro- 
duced, yet,  as  a  practical  consideration,  it  is  con- 
venient to  consider  at  least  two  types  of  staphy- 
lococci: the  Staphylococcus  pyogenes  aureus  and 
the  Staphylococcus  pyogenes  albus.  In  culturing 
from  the  pus  of  an  abscess  or  furuncle  we  generally 
obtain  a  golden  coccus,  while  in  material  from  the 
nose  or  mouth,  the  staphylococcus  colonies  are 
almost  invariably  white.  As  regards  the  common 
skin  coccus,  this  will  be  found  to  produce  a  white 
colony.  A  coccus  which  very  slowly  liquefies  gelatin  and  has  been 
supposed  to  cause  stitch  abscesses  is  the  S.  epidermidis  albus. 

While  it  is  customary  to  look  for  a  golden  colony  in  the  case  of  organisms  show- 
ing virulence,  yet  at  times  a  cream-white  colony  may  develop  from  cocci  of  great 
virulence. 

The  S.  pyogenes  citreus  is  considered  as  of  very  feeble  pathogenic  power.  Cer- 
tain cocci  whose  colonies  have  presented  a  waxy  appearence  have  been  designated 
as  S.  cereus  albus  and  S.  cereus  flavus,  respectively.  They  are  of  very  little  practi- 


FIG.  12. — Gektine 
culture  Staphylococ- 
cus aureus  one  week 
old.  (Williams.) 


THE   PNEUMOCOCCUS  55 

cal  importance.  The  Staphylococcus  pyogenes  aureus  grows  readily  at  room  tempera- 
ture, but  better  at  37°  C.  It  coagulates  milk  and  renders  bouillon  uniformly  turbid. 
It  grows  on  all  media,  as  blood-serum,  agar,  potato,  etc.  It  has  been  proposed  to 
distinguish  it  from  skin  staphylococci  by  its  power  of  producing  acid  in  mannite. 
Ordinarily  the  individual  cocci  are  about  i//  in  diameter,  but  they  vary  greatly  in 
size  according  to  the  age  of  the  culture  and  other  conditions.  The  aureus,  as  it 
is  frequently  called,  is  not  only  often  found  in  circumscribed  processes,  but  it  is  a 
frequent  cause  of  septicaemia,  osteomyelitis,  endocarditis,  etc. 

In  infection  of  bone  tissue  the  Staphylococcus  is  by  far  the  most  frequent  cause. 
It  is  well  to  remember  that  insignificant  staphylococcal  infection  may  lead  to  sep- 
ticaemia. In  the  tropics,  where  resistance  is  often  lowered  and  staphylococcal  skin 
infections  common,  continued  fevers  are  often  septicaemias.  It  is  the  organism 
most  frequently  concerned  in  terminal  infections.  The  lowered  resistance  of  the 
patient  permits  of  their  passage  through  barriers  ordinarily  resistant.  Not  only 
should  this  be  kept  in  mind  when  such  organisms  are  isolated  at  an  autopsy,  but 
as  well  the  fact  that  their  entrance  may  have  been  agonal  or  subsequent  to  death. 

The  Pneumococcus  of  Fraenkel. — (Weichselbaum  differentiated 
organisms  causing  pneumonia  in  1886.)  This  is  by  far  the  most  com- 
mon cause  of  pneumonia,  whether  it  be  of  the  croupous,  catarrhal,  or 
septic  type.  It  is  also  frequently  found  in  meningitis,  empyema,  endo- 
carditis and  otitis  media.  It  should  not  be  confused  with  the  pneumo- 
bacillus  of  Friedlander,  which,  although  possessing  a  capsule  like  the 
pneumococcus,  differs  from  it  by  being  Gram  negative,  being  a  bacillus 
and  having  large  viscid  colonies.  The  pneumococcus  is  the  cause  of 
more  than  80%  of  the  cases  of  pneumonia.  It  does  not  grow  below 
20°  C.  and  is  best  cultivated  on  blood-serum,  or  blood-streaked  agar. 
On  plain  agar  it  grows  as  a  very  small  dew-drop-like  colony,  which  is 
slightly  grayish  by  reflected  light.  It  produces  considerable  acid,  thus 
acidifying  and  usually  coagulating  litmus  milk.  It  produces  acid  in 
inulin  media  which  the  streptococcus  fails  to  do.  The  colony  is  smaller 
and  more  transparent  than  a  streptococcus  colony.  In  sputum  or 
other  pathological  material  it  is  best  recognized  by  the  presence  of  a 
capsule  inclosed  in  which  are  two  lance-shaped  cocci  with  their  bases 
apposed.  In  artificial  culture  we  rarely  get  the  capsule.  It  also  some- 
times grows  in  short  chains  like  a  streptococcus.  The* best  medium 
for  differentiating  is  the  serum  of  a  young  rabbit;  in  this  it  grows  as  a 
diplococcus,  while  streptococci  show  chains.  The  best  method  of 
isolating  it  in  pure  culture  is  to  inject  the  sputum  into  the  marginal 
ear  vein  of  a  rabbit  or  subcutaneously  into  a  mouse.  Death  results 
from  septicaemia  in  about  two  days  and  the  blood  teems  with  pneu- 
mococci.  Usually  the  pneumococcus  quickly  loses  its  virulence,  and 


56  STUDY   AND    IDENTIFICATION   OF  BACTERIA 

also  dies  out  in  a  few  days  unless  transferred  to  fresh  media.  The 
best  medium  for  its  preservation  is  rabbit's  blood  agar;  this  also  main- 
tains the  virulence.  On  this  medium  the  colonies  are  larger  than  on 
agar  and  they  present  a  greenish  appearance. 

The  pneumococcus  growth  emulsifies  very  readily  and  evenly  so  that  suspensions 
for  vaccines  are  easily  made. 

It  is  a  well-known  fact  that  the  pneumococcus  is  a  frequent  inhabitant  of  the 
nasal,  pharyngeal,  and  buccal  cavities.  The  explanation  of  infection  is  either  on 
the  ground  of  lowered  resistance  of  the  patient  or  enhanced  virulence  of  the  organism. 
Oscar  Richardson  has  reported  an  organism  in  cases  of  lobar  pneumonia,  cerebro- 
spinal  meningitis,  mastoid  disease,  etc.,  bearing  resemblance  to  both  pneumococci 
and  streptococci — the  Streptococcus  capsulatus.  It  differs  from  the  pneumococcus 


**•£•*«  *;. 

*f  T*k~i 

•«*  '       ds* 


r 

•  •m.m^*^-'*jf 


FIG.  13. — Pneumococcus,  showing  capsule,  from  pleuritic  fluid  of  infected  rabbit, 
stained  by  second  method  of  Hiss.     (Williams.) 

in  that  the  colonies  on  blood-serum  are  viscid  and  like  irregular  flecks  of  mucus. 
The  characteristic  culture  is  a  glucose  agar  stab.  (Reaction  must  not  exceed  +0.5.) 
From  the  line  of  puncture  there  are  flail-like  projections  extending  outward  from 
one-fifth  to  one-fourth  of  an  inch.  The  capsule  persists  on  ordinary  culture  media. 
This  organism  resembles  the  Streptococcus  of  Bonome  of  the  French. 

In  a  study  of  blood  and  sputum  cultures  from  thirty- two  cases  of  lobar 
pneumonia  Hastings  and  Boehm  found  blood  and  sputum  positive  bacteriologically 
in  eleven  cases.  In  nine  of  these  cases  the  pneumococcus  was  isolated  and  in 
two  a  haemolysing  streptococcus.  In  the  other  twenty-one  cases  the  sputum 
cultures  were  bacteriologically  positive  in  eighteen  of  the  cases  and  negative  in 
three.  In  nine  cases  the  pneumococcus  was  isolated,  in  two  cases  B.  coli,  in 
one  case  M.  catarrhalis,  in  one  case  a  staphylococcus,  in  two  cases  staphylococci 
and  streptococci,  in  one  case  B.  influenzas.  The  percentage  of  positive  blood 


GONOCOCCUS  57 

cultures  was  30.3.     Cole  obtained  30%  of  positive  blood  cultures.     The  blood  was 
taken  into  flasks  of  bouillon  in  dilution  of  1-50. 

Diplococcus  crassus. — This  is  a  Gram  positive,  kidney-shaped 
diplococcus,  which  might  be  confused  with  the  M.  catarrhalis  or  the 
meningococcus  by  ordinary  staining  methods.  It  is  larger  than  the 
meningococcus. 

In  throat  cultures  I  have  isolated  on  several  occasions  a  Gram  positive  diplococcus 
which  is  at  times  biscuit-shaped,  at  times  irregularly  spherical.  It  possesses  two 
or  three  metachromatic  granules,  so  that  in  a  Neisser  stain  for  diphtheria  the  appear- 
ance of  these  granules  may  be  confusing. 

Using  Ponder's  toluidin  blue  stain  I  have  observed  granule  staining  in  organisms 
of  round  or  oval  morphology  which  were  suggestive  of  the  ascospore  staining  of 
yeasts. 

Gram  Negative  Cocci. — It  is  important  to  bear  in  mind  that  there 
are  many  cocci  of  varying  shapes,  which  in  cultures  or  in  smears  from 
the  throat,  nose  or  faeces  are  Gram  negative.  These  are  not  well  classi- 
fied or  described.  To  distinguish  the  three  important  kidney-shaped 
diplococci,  it  can  be  most  easily  accomplished  by  cultural  methods, 
using  hydrocele  agar  (ascites  or  blood  agar  will  answer),  ordinary  blood- 
serum  and  plain  agar.  The  gonococcus  will  only  grow  on  the  hydro- 
cele agar;  the  meningococcus  will  grow  on  this,  but  likewise  grows  on 
ordinary  blood-serum.  The  M.  catarrhalis  will  grow  on  plain  agar  as 
well  as  on  other  media. 

Other  Gram  negative  organisms  of  confusing  morphology  are  M. 
pharyngis  siccus,  the  colonies  of  which  show  great  crinkly  dryness,  and 
M.  pharyngis  flavus. 

Gonococcus  (Neisser,  1879). — This  organism  is  characteristically 
a  diplococcus,  the  separate  cocci  being  plano-convex  with  their  plane 
surfaces  apposed.  (Biscuit  shape,  coffee-bean  shape.)  They  are  gen- 
erally found  grouped  in  masses  of  several  pairs,  most  strikingly  in  pus 
cells  or  epithelial  cells,  but  also  found  extracellularly.  Except  in  the 
height  of  the  disease,  there  is  a  great  tendency  for  the  organisms  to  show 
involution  forms,  so  that  instead  of  biscuit-shaped  diplococci  we  have 
round,  irregular  and  uneven  cocci.  It  is  therefore  advisable  in  search- 
ing smears  from  chronic  gonorrhoea  to  continue  the  search  of  Gram- 
stained  specimens  until  some  fairly  typical  diplococci  are  found.  There 
is  nothing  requiring  greater  discrimination  than  a  diagnosis  from  such 
a  smear.  At  the  commencement  of  a  gonorrhoea  the  epithelial  cells  are 
abundant  and  gonococci  are  found  adhering  to  them  or  lying  free. 


58  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

Later  on,  at  the  acme  of  the  discharge  (the  creamy,  abundant  discharge) , 
it  is  in  the  pus  cells  we  find  them  and  they  may  be  so  abundant  that  10 
to  20%  of  the  pus  cells  may  contain  them.  In  the  subacute  stage  the 
epithelial  cells,  which  practically  disappear  when  the  discharge  is  so 
abundant,  begin  to  reappear,  and  in  the  chronic  stage  the  epithelial 
cells  are  the  chief  ones,  and  are  the  ones,  on  which  we  find  an  occasional 
gonococcus,  often  distorted  in  shape. 

The  best  method  of  diagnosis  in  cases  of  chronic  gonorrhoea  is  to  have  the  patient 
drink  beer  and  eat  the  stimulating  food  previously  interdicted,  to  take  active  ex- 
ercise and  to  have  a  sound  passed.  To  obtain  material  for  examination  ihe  glans 
penis  should  be  washed  and  the  patient  who  has  presented  himself  with  a  full 
bladder  should  pass  a  portion  of  the  contained  urine.  Next  the  prostate  aj*9  seminal 


FIG.  14. — Gonococcus.     Film  from  urethral  pus.     (Coplin.) 

vesicles  should  be  massaged  with  the  patient  standing  but  bent  over  and  the  penis 
pendant.  The  drops  of  discharge  from  the  massage  should  be  received  in  a  small 
Petri  dish  and  finally  the  remaining  urine  should  be  passed  into  a  sterile  bottle. 
Smears  and  cultures  should  be  made  from  the  sediment  of  the  two  urinary  specimens 
and  from  the  secretions  of  the  massaged  prostate  and  vesicles. 

The  smears  made  from  the  resulting  discharge  or  centrifuged  urine  will  probably 
contain  gonoccocci  if  they  are  present  in  the  urethra.  In  the  female  the  favorite 
sites  are  the  urethra  and  the  cervix  uteri.  In  municipal  examinations  it  is  customary 
to  make  two  smears:  one  from  the  urethral  meatus  and  a  second  from  the  cervix. 
The  vagina  is  not  a  suitable  soil  for  their  development.  In  female  children  it  is 
most  often  found  in  the  discharge  of  the  vulvovaginitis. 


MENINGOCOCCUS  59 

In  addition  to  the  genital  organs,  the  gonococcus  may  at  times  invade  and  be 
isolated  from  the  eye  (gonorrhoeal  ophthalmia),  the  joints,  rarely  as  a  cause  of 
endocarditis  and  possibly  as  the  factor  in  septicaemia.  Grown  upon  hydrocele  or 
ascites  agar,  or  blood-streaked  agar,  or  upon  blood  agar  from  man  or  the  rabbit, 
the  colonies  appear  as  irregular,  minute,  dew-drop  spots.  By  the  second  or  third 
day  the  involution  forms  are  abundant,  and  within  four  to  seven  days  the  culture 
will  probably  be  found  to  be  dead.  Unless  frequent  transfers  are  made,  it  will  be 
best  kept  alive  on  blood  agar.  The  organism  grows  best  at  37°  C.,  and  will  not 
grow  below  25°  C.  It  will  not  grow  on  plain  or  glycerine  agar  or  ordinary  blood- 
serum  unless  the  transfer  of  considerable  pus  in  inoculating  the  slants  gives  it  a 
suitable  culture  medium.  In  material  from  joints,  it  is  in  the  fibrin  flakes  that  the 
gonococci  are  most  .apt  to  be  found,  if  found  at  all. 

By  heating  the  blood-streaked  agar  tubes  to  56°  C.  for  twenty  minutes  (in- 
activation-destroying  complement  and  hence  bactericidal  power  of  blood  on  slant) 
greater  success  in  primary  cultures  will  be  obtained. 

In  culturing  gonococci  the  transfer  of  material  to  culture  media 
should  be  made  with  the  least  delay  possible. 

The  most  satisfactory  medium  is  Thalman's  medium  upon  the 
slanting  surface  of  which  we  have  deposited  two  or  three  drops  of 
human  serum.  Blood  may  be  taken  from  a  vein  or  the  Wright  U  tube 
may  be  used  and  after  centrifuging  the  sterile  serum  is  taken  off  with 
a  capillary  bulb  pipette  and  deposited  on  and  smeared  out  on  the  slant. 

Diplococcus  intracellularis  meningitidis  (Weichselbaum,  1887). — 
This  is  the  organism  of  epidemic  cerebrospinal  meningitis,  and  is 
frequently  termed  the  meningococcus.  The  diplococcus  is  Gram 
negative  and  biscuit-shaped  and  is,  like  the  gonococcus,  chiefly  con- 
tained in  pus  cells.  It  is  also  found  free  in  the  cerebrospinal  fluid 
withdrawn  from  cerebrospinal  fever  cases.  There  is  a  greater  tendency 
to  variation  in  size  and  shape  than  is  the  case  with  the  gonococcus, 
which  latter,  in  fresh  material,  shows  a  striking  uniformity  morpholog- 
ically. The  meningococcus  is  at  times  not  abundant— early  in  the  case, 
however,  the  picture  may  be  similar  to  that  of  gonorrhoea. 

On  blood-serum  the  colonies  appear  after  twenty-four  to  forty-eight  hours  as 
discrete,  very  slightly  hazy  colonies,  about  one-tenth  of  an  inch  in  diameter.  On 
serum  agar,  as  ascites  or  hydrocele  agar,  they  grow  best.  Unless  considerable 
cerebrospinal  fluid  is  transferred  with  the  inoculating  loop,  they  do  not  grow  on 
plain  agar.  They  will  grow  at  times  on  glycerine  agar.  The  organism  is  very 
sensitive  to  light,  cold  and  drying.  It  ferments  dextrose  and  only  grows  at  blood 
temperature,  thus  distinguishing  it  from  the  M.  catarrhalis.  It  is  scarcely  patho- 
genic for  laboratory  animals,  with  the  exception  of  the  mouse  and  guinea-pig, 
when  intraperitoneal  injections  but  not  subcutaneous  ones  give  results.  Intradural 
injections  give  results.  The  cultures  die  out  very  rapidly,  so  that  it  is  necessary  to 


60  STUDY   AND    IDENTIFICATION   OF  BACTERIA 

make  transfers  every  one  or  two  days.  The  meningococcus  has  been  isolated  from 
the  nasal  secretions  of  patients.  The  possibility  of  these  organisms  being  the  M. 
catarrhalis  must  be  considered. 

Flexner  has  shown  that  in  monkeys,  which  are  susceptible  to  the  disease,  in- 
jections of  cultures  of  M.  intracellularis  into  the  spinal  canal  is  followed  by  migra- 
tion of  the  cocci  to  the  nasal  cavity  both  free  and  in  phagocytic  leukocytes. 

The  meningococcus  has  a  very  slight  resistance  to  sun  or  drying  so 
that  its  aerial  transmission  seems  doubtful.  It  is  supposed  to  effect 
an  entrance  by  the  nares,  thence  reaching  the  cerebral  meninges.  Inf  ec- 


FIG.  15. — Diplococcus  intracellularis  meningitidis  and  pus  cells.  (Xiooo.) 

(Williams.} 

tion  is  probably  by  direct  contagion.  Several  cases  have  been  re- 
ported where  with  a  high  leukocytosis  the  cocci  have  been  found  in  the 
polymorphonuclears  of  blood  smears  and  in  cultures  from  the  blood. 
(In  about  25%  of  blood  cultures  where  from  5  to  10  c.c.  are  employed.) 

By  the  use  of  initial  injections  into  horses  of  killed  cultures  followed  by  alternate 
injections  into  horses  of  living  diplococci,  then  seven  days  later  of  an  autolysate 
made  from  different  strains;  seven  days  later  again  injecting  living  diplococci;  thus 
alternating  material  every  week,  an  antiserum  of  value  has  been  obtained  by  Flexner. 
The  immunization  requires  about  one  year.  In  using,  withdraw  about  20  c.c.  of 
patient's  cerebrospinal  fluid  with  a  syringe,  and  then  inject,  through  the  same  needle, 
an  equal  quantity  of  the  serum.  The  injection  is  repeated  every  day  for  three  or 
four  days. 

For  diagnosis,  make  smears  and  cultures  from  cerebrospinal  fluid. 
The  sediment  from  the  centrifuged  material  gives  better  results.  In 
tuberculosis  the  lymphocytes  preponderate;  in  cerebrospinal  meningitis 
the  polymorphonuclears. 


MALTA  FEVER  6 1 

It  has  been  stated  that  a  point  of  difference  between  the  phagocytosis  with  the 
gonoccoci  and  the  meningococci  is  that  the  meningococci  invade  and  at  times  destroy 
the  nucleus  of  the  polymorphonuclear,  which  is  not  true  of  gonococci.  The  appear- 
ance of  large  phagocytic  endothelial  cells,  often  containing  polymorphonuclears,  in 
the  centrifuged  cerebrospinal  fluid  is  a  favorable  prognostic  sign.  At  times  there 
does  not  appear  to  be  any  relation  between  the  number  of  phagocytic  polymorpho- 
nuclears and  the  severity  of  the  case. 

Vincent  has  recommended  a  precipitin  test  for  epidemic  cerebrospinal  meningitis 
which  has  the  advantages  of  being  simple  and  more  immediate  than  cultures  and  of 
particular  value  in  those  cases  when  meningococci  cannot  be  found  in  the  smears  or 
in  cultures  from  the  cerebrospinal  fluid.  It  is  performed  by  adding  one  or  two  drops 
of  antimeningococcic  serum  to  a  tube  of  fresh  cerebrospinal  fluid  which  has  been 
cleared  by  centrifugalization  for  10  to  15  minutes.  After  adding  the  serum  the  tube 
is  placed  in  the  incubator  at  52°  C.  for  two  to  five  hours  together  with  a  control 
tube.  The  formation  of  a  precipitate  (turbidity)  shows  a  postive  test. 

Micrococcus  catarrhalis  (Seifert,  1890). — This  organism  has  been 
specially  studied  by  Lord.  It  resembles  the  meningococcus  strikingly 
and  can  only  be  differentiated  by  cultural  procedures.  It  grows  on  plain 
agar  and  at  room  temperature,  and  does  not  produce  acid  in  glucose 
media.  It  not  only  occurs  in  the  nasal  secretions  of  healthy  people, 
but  appears  to  be  responsible  for  certain  coryzas  and  bronchial  affec- 
tions, resembling  influenza.  It  also  is  responsible  for  certain  epidemics 
of  conjunctivitis. 

The  original  cultures  may  show  only  slight  growth  whereas  the  sub- 
cultures prove  luxuriant. 

The  colonies  are  larger,  more  opaque,  and  have  a  more  irregular 
wavy  border  than  the  round  colonies  of  the  meningococcus. 

Micrococcus  melitensis  (Bruce,  1887). — This  is  the  organism  of 
Malta  or  Mediterranean  fever,  sometimes  called  undulant  fever,  on 
account  of  successive  waves  'of  pyrexia  running  over  several  months. 
The  disease  has  a  very  slight  mortality  (2%),  and  the  lesions  are  chiefly 
of  the  spleen,  which  is  large  and  diffluent.  The  organisms  can  best  be 
isolated  from  the  spleen. 

M.  melitensis  is  only  about  0.3/1  in  diameter.  The  characteristics  are  its  very 
small  size  and  the  dew-drop  minute  colonies  on  agar,  which  at  incubator  temperature 
only  show  themselves  about  the  third  to  the  sixth  day.  It  is  nonmotile  and  Gram 
negative.  In  bouillon  there  is  a  slight  turbidity. 

Many  laboratory  infections  have  been  recorded. 

The  organism  occurs  in  peripheral  circulation,  it  having  been  cultivated  from 
blood  very  successfully  by  Eyre.  He  takes  blood  at  the  height  of  the  fever,  and  in 
the  afternoon.  Formerly  it  was  customary  to  isolate  by  splenic  puncture. 

Infection  is  chiefly  by  means  of  the  milk  of  infected  goats.     The  organisms  are 


62  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

excreted  in  the  urine  of  patients,  and  a  diagnostic  point  is  to  make  plates  from  the 
urine.  Such  urine  applied  to  abraded  surfaces  causes  infection. 

The  serum  of  patients  shows  agglutinating  power  as  early  as  the  fifth  day  of 
the  disease,  and  this  may  persist  for  years  after  recovery.  Nicolle  has  advised  using 
serum  heated  to  56°  C.  for  30  minutes  for  the  agglutination  test,  nonspecific  agglu- 
tinins  being  thereby  destroyed.  Carriers  may  be  of  importance  in  Malta  fever  and 
are  best  detected  by  agglutination  tests. 

A  high  mononuclear  increase  may  be  found  in  this  disease. 


CHAPTER  VI. 

STUDY  AND   IDENTIFICATION   OF  BACTERIA.     SPORE- 
BEARING  BACILLI.    KEY  AND  NOTES. 

A.  Grow  aerobically. 

1.  Stab  culture  in  gelatin  has  branches  growing  out  at  right  angles  to  line  of  stab. 

a.  Has  no  membrane  on  bouillon  or  liquefied  gelatin.     Projecting  branches 
from  line  of  stab  only  at  upper  part  of  line  of  growth.     Absolutely  nonmotile, 
Ends  sharply  cut  across  or  concave.     ANTHRAX  GROUP. 

b.  Has  thick  whitish  membrane  on  bouillon  and  surface  of  liquified  gelatin. 
Projecting  branches  all  along  the  line  of  stab.     Sluggishly  motile.     MY- 
COIDES  GROUP.     (B.  mycoides.     B.  ramosus.) 

2.  Stab  cultures  in  gelatin  do  not  show  projecting  branches. 

a.  Potato  cultures  do  not  become  wrinkled.     At  first  slightly  moist,  later 
dry    and   mealy.     SUBTILIS    GROUP.     (Hay   bacillus   group.)     Actively 
motile  with  more  or  less  square  ends  and  a  central  spore  which  is  of  the  same 
diameter  or  only  slightly  larger  than  the  bacillus.     The  yellow  subtilis  is  at 
times  found  in  water.     The  colonies  on  potato  are  of  a  cheese-yellow  color. 
The  bacilli  are  very  large  and  show  a  sluggish,  worm-like  motion. 

B.  megatherium  often  shows  a  granular  or  beaded  appearance  in  a  Gram 
preparation.  The  narrow  spores  are  never  central,  usually  between  center 
and  end,  and  rather  elongated.  It  most  nearly  resembles  the  sporulating 
bacillus  of  malignant  oedema  but  if  the  spore  is  quite  terminal  and  bulging 
may  resemble  B.  tetani. 
Cultures  of  B.  megatherium  are  somewhat  similar  to  B.  coli  colonies. 

b.  Potato  cultures  at  first  even  growth  but  after  a  few  days  become  wrinkled. 
VULGATUS  GROUP.     (Potato  bacillus.) 

B.  vulgatus  shows  marked  wrinkling,  like  intestinal  coils.     B.  mesentericus 
show  slight  wrinkling  and  a  network-like  appearance. 

Two  water  bacilli  belonging  to  this  group  are  the  B.  mesentericus  fuscus 
(brown  growth)  and  B.  mesentericus  ruber  (red  growth). 

NOTE. — The  following  cultural  characteristics  are  common  to  all  the  above  spore 
bearers. 

1.  Liquefaction  of  gelatin. 

2.  Milk  slowly  and  incompletely  coagulated  with  very  little  change  in  reaction. 
Later  the  coagulum  is  digested. 

3.  No  gas  in  either  glucose  or  lactose. 

4.  No  indol. 

5.  All  are  Gram  positive. 

6.  All  digest  blood-serum. 

63 


64  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

B.  Grow  only  anaerobically. 

1.  Rods  very  little  swollen  by  centrally  situated  spores. 

a.  Motile.     B.  cedematis  maligni.     (Gram  negative.) 

b.  Non  motile.     B.  aerogenes  capsulatus.     (Capsule.) 

2.  Spores  tend  to  be  situated  between  center  and  end. 

a.  No  liquefaction  of  gelatin.     B.  butyricus. 

b.  Gelatin  liquefied  slowly. 

B.  botulinus.     Milk  not  coagulated. 

B.  anthracis  symptomatici. 

B.  enteritidis  sporogenes.     Milk  coagulated  with  abundant  gas. 

c.  Gelatin  liquefied  rapidly.     B.    cadaveris  sporogenes.     Very  motile. 

3.  Spores  situated  at  end  of  rod.     Drum-stick  sporulation.     TETANUS  GROUP. 

The  following  table  taken  from  Lehmann  and  Neumann,  based 
on  pathogenic  effects,  is  of  great  practical  value.  After  inoculation 
of  some  animal  subcutaneously  with  the  suspected  material  we  have: 

A.  No  particular  symptoms  at  site  of  inoculation. 
Absorption  of  the  soluble  toxin  causing: 

(1)  General  symptoms  of  tetanus.     B.  tetani. 

(2)  Botulism  poisoning   symptoms.     Pupillary  symptoms.     Paralysis  of 
tongue  and  pharynx.     Cardiac  and  respiratory  failure. 

B.  Local  symptoms  marked  at  site  of  inoculation.     Hemorrhagic  emphysematous 
oedema. 

(1)  Motile. 

(a)  Gram  negative.  B.  cedematis  maligni. 

(b)  Gram  positive.  B.  anthracis  symptomatici. 

(2)  Nonmotile. 

B.  aerogenes  capsulatus  or  B.  phlegmonis  emphysematosse. 

SPORE-BEARING  AEROBES. 

Bacillus  anthracis  (Pollender  discovered  1849.  Davaine  recog- 
nized nature  1863.  Koch  proved  1876). — Of  the  aerobic  spore-bearing 
bacilli  this  is  the  only  one  of  particular  medical  importance. 

Anthrax  is  an  important  disease  in  domestic  animals,  especially 
sheep  and  cattle.  The  characteristic  postmortem  change  in  animals 
is  the  greatly  enlarged,  friable,  mushy  spleen.  Man  is  much  less  suscep- 
tible than  these  animals,  but  is  more  so  than  the  goat,  horse,  or  pig. 
The  Algerian  sheep  has  a  high  degree  of  immunity,  as  has  the  white  rat. 
The  brown  rat  is  quite  susceptible.  The  disease  in  man  chiefly  occurs 
among  those  working  with  hides,  wool,  or  meat  of  infected  cattle.  The 
two  chief  types  in  man  are:  i.  Malignant  pustule  and  2.  Woolsorter's 
disease.  An  intestinal  type  is  also  recognized.  Malignant  pustule 


ANTHRAX  65 

results  from  the  inoculation  of  an  abrasion  or  cut;  thus  it  frequently 
shows  on  the  arms  and  the  backs  of  those  unloading  hides.  It  first 
appears  as  a  pimple,  the  center  of  which  becomes  vesicular,  then 
necrotic. 


FIG.  1 6. — Anthrax  bacilli.     Cover-glass  has  been  pressed  on  a  colony  and  then  fixed 
and  stained.     (Kolle  and  Wasscrmann.) 


FIG.  17. — Anthrax  bacilli  growing  in  a  chain  and  exhibiting  spores. 
(Kolle  and  Wasserman.) 

A  ring  of  vesicles  surrounds  this  central  eschar  and  a  zone  of  congestion,  the 
vesicles.  The  lymphatics  soon  become  inflamed  as  well  as  neighboring  glands. 
If  the^mstule  is  not  excised  and  death  occurs,  there  is  not  much  enlargement  of  the 
spleen  and  the  bacteria  are  not  abundant  in  the  kidneys,  etc.,  as  with  animals. 
Man  seems  to  die  from  a  toxaemia  rather  than  a  septicaemia. 

In  woolsorter's  disease  there  is  great  swelling  and  oedema  of  the  bronchial  and 
mediastinal  glands.  The  lungs  show  oedema,  which  about  the  bronchi  is  hemorrhagic. 

5 


66  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

The  bacillus  is  5  to  8/*  by  i  to  i  i/2//.  It  has  square  cut  or  concave 
ends  and  is  often  found  in  chains.  It  is  Gram  positive.  Colonies,  by 
interlacing  waves  of  strings  of  bacteria,  show  Medusa  head  appearance. 
For  cultural  characteristics  see  key.  Spores  develop  best  at  a  tempera- 
ture of  30°  C.  They  stain  with  difficulty. 

Stiles  thinks  that  animals  are  infected  by  eating  the  bones  of  animals  which  have 
died  of  anthrax,  cutting  buccal  mucous  membrane,  and  so  becoming  infected. 
Spores  do  not  form  in  an  intact  animal  body,  but  they  do  form  after  a  postmortem 
or  the  disintegration  of  the  body  by  maggots.  For  this  reason  it  is  better  not  to 
open  up  the  body  of  the  animal,  but  to  make  the  diagnosis  by  cutting  off  an  ear. 
Dried  spores  will  live  for  years  and  will  withstand  boiling  temperature  for  hours. 

In  vaccinating  animals  against  anthrax,  Pasteur  used  two  vaccines.  The  first 
is  attenuated  fifteen  days  at  42.5°  C.  The  second,  attenuated  for  only  ten  days,  is 
given  twelve  days  later. 


FIG.  18. — Bacillus  anthracis  in  blood  of  rabbit.     (Coplin.} 

In  taking  material  from  a  malignant  pustule  before  excision,  be 
careful  not  to  manipulate  it  roughly,  as  bacteria  may  enter  the  circula- 
tion. Make  cover-glass  preparations,  staining  by  Gram.  Make 
culture  on  agar.  Blood  cultures  are  usually  only  positive  later  in  the 
disease.  Inoculate  a  guinea-pig  or  a  mouse  subcutaneously. 

The  guinea-pig  dies  in  about  forty-eight  hours  and  shows  an  cedematous  gelatin- 
ous exudate  at  site  of  inoculation.  The  blood  is  black  and  swarms  with  anthrax 
bacilli.  It  is  the  best  example  of  a  septicaemia. 

An  organism  with  a  central  spore  and  morphologically  resembling 
B.  anthracis,  but  motile,  has  been  reported  as  occurring  in  the  stools 
of  pellagrins.  Gelatine  stabs  show  a  cup-shaped  liquefaction  in  about 


SPORE  BEARING   ANAEROBES 


67 


five  days.  No  change  in  milk.  The  colonies  are  slimy  and  opaque. 
The  organism  is  said  to  be  agglutinated  by  the  serum  of  pellagra  cases. 
The  name  B.  MAYDIS  has  been  given  to  it. 

SPORE-BEARING  ANAEROBES. 

There  are  three  very  important  pathogens  in  this  group — that  of 
malignant  oedema;  that  of  botulism,  and  the  organism  of  tetanus. 

The  B.  enteritidis  sporogenes  is  of  importance  in  connection  with 
indications  of  faecal  contamination  of  water.  In  connection  with  B. 
aerogenes  capsulatus,  there  is  some 
question  as  to  whether  the  exten- 
sive oedema  produced  by  it  may 
not  usually  be  from  a  terminal  or 
cadaveric  infection.  At  any  rate 
necrotic  material  seems  necessary. 

It  should  be  stated  that  our 
knowledge  of  the  differential  cul- 
tural characteristics  of  anaerobes 
is  unsatisfactory.  The  exact 
methods  which  are  in  use  for 
aerobes  have  not  been  applied 
to  anaerobic  organisms. 

To  Cultivate  Anaerobes. — 
Probably  the  apparatus  giving 
the  most  perfect  anaerobic  con- 
ditions is  the  Novy  jar,  in  which 
the  air  has  been  replaced  by 
hydrogen.  The  difficulties  attending  the  method  are: 


FIG.  19. — Novy  jar. 


1.  Unless  a  special  apparatus  (Kipp's)  is  at  hand,  there  may  be  difficulty  in  pre- 
venting the  sulphuric  acid  from  frothing  over  when  poured  on  the  zinc.     It 
should,  at  first,  be  added  in  small  quantities  at  a  time — well  diluted  (i  to  6). 

2.  Various  wash-bottles  are  required:  one  containing  silver  nitrate  solution  for 
traces  of  AsHs  and  one  with  lead  acetate  for  H^S  and  another  with  pyrogallic 
acid  and  caustic  soda  for  any  oxygen  that  may  come  over. 

3.  Mixtures  of  hydrogen  and  air  explode.     Consequently,  in  determining  whether 
all  air  has  been  expelled  and  in  its  place  an  atmosphere  of  hydrogen  exists,  it 

is  necessary  to  see  if  the  escaping  gas  burns  with  a  blue  flame.     Unless  this 
is  collected  in  a  test-tube  and  examined,  we  may  have  an  explosion. 

4.  Except  in  a  large  laboratory,  where  the  apparatus  is  set  up  and  ready  for  use, 
too  much  time  would  be  required. 

5.  Simpler  methods  appear  to  give  as  good  results. 


68 


STUDY   AND    IDENTIFICATION   OF   BACTERIA 


In  Tarozzi's  method,  pieces  of  fresh  sterile  organs  are  added  to 
bouillon.  Pieces  of  kidney,  liver,  or  spleen  are  best  suited.  After 
adding  the  tissue  the  media  may  be  heated  to  80°  C.  for  a  few  minutes 
without  interfering  with  the  anaerobic  condition  producing  properties 
of  the  fresh  tissues.  This  method  is  practically  the  same  as  that 
recommended  by  Smith  (see  Tetanus).  This  is  also  a  feature  of 
Noguchi's  method  of  culturing  Treponema  pallidum, 

The  Method  of  Liborius. 

In  this  it  is  necessary  to  have  a  test-tube  containing  about  4  inches  of  a  i% 
glucose  agar.  Glucose  acts  as  a  reducing  agent  and  furnishes  energy.  It  is  con- 
venient to  add  about  i/io  of  i%  of  sulphindigo- 
tate  of  soda;  the  loss  of  the  blue  color  at  the  site 
of  the  colony  enabling  us  to  pick  them  out.  The 
tube  of  agar  should  be  boiled  just  before  using  to 
expel  remaining  oxygen  from  the  tube.  Now 
rapidly  bring  down  the  temperature  to  about  42° 
C.,  by  placing  the  tube  in  cold  water,  and  inocu- 
late the  material  to  be  examined.  A  second  or 
third  tube  may  be  inoculated  from  the  first,  just 
as  in  ordinary  diluting  methods  for  plate  cultures. 
Having  inoculated  the  tubes,  solidify  them  as 
quickly  as  possible,  using  tap  water  or  ice-water. 
The  anaerobic  growth  develops  in  the  depths  of 
the  medium.  Some  pour  a  little  sterile  vaseline 
or  paraffin  or  additional  agar  on  the  top  of  the 
medium  in  the  tube  as  a  seal  'from  the  air. 
Others  have  recommended  the  inoculation  of 
some  aerobe,  as  B.  prodigiosus,  on  the  surface. 
This  latter  method  is  not  advisable.  A  deep  stab 
culture  is  often  sufficient. 

The  Method  of  Buchner. 

In  this  method  one  gram  each  of  pyrogallic 
acid  and  caustic  potash  or  soda  for  every  100  c.c. 
of  space  in  the  vessel  containing  the  culture  is 
used  to  absorb  the  oxygen.  It  is  convenient  to 
drop  in  the  pyrogallic  acid;  then  put  in  place  the 
inoculated  tubes  or  plates;  then  quickly  pouring 
in  the  amount  of  caustic  soda,  in  a  10%  aqueous 
solution,  to  immediately  close  the  containing 
vessel.  A  large  test-tube  in  which  a  smaller  one  containing  the  inoculated  medium 
is  placed,  and  which  may  be  closed  by  a  rubber  stopper,  is  very  convenient.  A 
good  rubber-band  fruit  jar  is  satisfactory.  A  desiccator  may  be  used  for  plates. 


FIG.  20. — Arrangement  of 
tubes  for  cultivation  of  anae- 
robes by  Buchner's  method. 

(Williams.) 


MALIGNANT   (EDEMA  69 

An  excellent  method  for  anaerobic  plates,  either  in  a  desiccator  with  the  pyrogallic 
acid  and  caustic  soda,  or  less  satisfactorily  in  the  open  air,  is  to  sterilize  the  parts 
of  the  Petri  dish  inverted;  that  is,  the  smaller  part  is  put  bottom  downward  in 
the  inverted  cover  (as  one  would  set  one  tumbler  in  another).  Then,  in  using, 
unwrap  the  Petri  dish,  lift  up  the  inner  part,  pour  in  the  inoculated  medium 
into  the  upturned  cover.  Then  immediately  press  down  the  inner  dish,  spreading 
out  a  thin  film  of  the  medium  between  the  two  bottoms. 

J.  H.  Wright's  Method. 

Make  a  deep  stab  culture  in  glucose  agar  or  gelatin,  preferably  boiling  the  media 
before  inoculating.  Then  flame  the  cotton  plug  and  press  it  down  into  the  tube  so 
that  the  top  lies  about  three-fourths  of  an  inch  below  the  mouth  of  the  test-tube. 
Next  fill  in  about  one-fourth  of  an  inch  with  pyrogallic  acid;  then  add  2  or  3  c.c.  of 
a  10%  solution  of  caustic  soda,  and  quickly  insert  a  rubber  stopper.  This  method 
is  one  of  the  most  convenient  and  practical,  and  is  to  be  strongly  recommended. 

Method  of  Vignal. 

In  this  a  section  of  glass  tubing  (1/4  in.)  is  drawn  out  at  either  end,  as  in  making  a 
bacteriological  pipette,  with  a  mouth-piece  containing  a  cotton  plug.  The  liquid 
agar  or  gelatin  is  then  inoculated  and  the  medium  drawn  up  into  the  tube.  In  a 
very  small  flame  the  capillary  narrowings  are  sealed  off,  and  we  have  inside  the  tube 
very  satisfactory  anaerobic  conditions.  To  get  at  the  colonies,  file  a  place  on  the 
tube  and  break  at  this  point. 

To  obtain  material  for  examination  and  isolation  in  pure  culture  from  the  deep 
agar  stab-tube,  it  is  best  to  loosen  the  medium  at  the  sides  of  the  tube  with  a  heated 
platinum  spud  or  a  flattened  copper  wire.  Then  shake  the  mass  out  into  a  sterile 
Petri  dish.  It  is  dangerous  to  break  the  tubes  with  a  hammer  as  some  do. 

A  Combination  Method. 

Recently  as  shown  in  the  illustration  in  Fig.  7,  I  have  been  combining  various 
methods  so  that  very  satisfactory  anaerobic  conditions  are  obtained.  First,  a 
deep  agar  stab  of  freshly  sterilized  glucose  agar  is  made.  The  surface  of  this  is  then 
covered  with  sterile  paraffin  oil.  The  proper  amount  of  pyrogallic  acid  is  then 
deposited  in  a  salt  mouth  bottle.  The  rubber  stopper  with  the  glass  and  rubber 
tubing  is  then  firmly  pushed  in  and  connection  made  with  a  filter  pump. 

In  five  to  ten  minutes  almost  all  the  air  will  be  exhausted  when  the  Hofmann 
clamp  is  screwed  up  tight  and  the  bottle  disconnected  from  the  vacuum  pump. 
The  glass  tubing  end  is  then  inserted  into  a  graduate  holding  10%  caustic  soda 
solution,  the  Hofmann  clamp  unscrewed,  and  the  necessary  amount  of  caustic  soda 
having  been  run  in,  as  noted  under  Buchner  method,  we  again  close  the  screw  clamp 
and  incubate. 

B.  oedematis  maligni  (Pasteur,  1877). — This  is  the  vibrion  septique 
of  Pasteur.  It  is  found  in  garden  soil  and  in  street  sweepings.  It  is 


7O  STUDY   AND    IDENTIFICATION   OF  BACTERIA 

the  cause  of  an  acute  cellular  necrosis  attended  with  serous  sanguino- 
lent  exudation  and  with  more  or  less  emphysema.  The  organism 
only  becomes  generalized  in  the  blood  about  the  time  of  death  and 
postmortem.  Therefore,  it  is  not  a  septicaemia,  as  is  anthrax.  The 
bacillus  is  an  organism  about  the  size  of  anthrax  (y/Jt  by  0.8),  but  is  nar- 
rower and  does  not  have  the  same  square  cut  or  dimpled  ends.  Further- 
more, it  is  motile,  Gram  negative  and  an  anaerobe.  The  guinea-pig 
is  very  susceptible,  and  about  the  time  of  death  and  postmortem  there 
may  be  seen  long  flexile  motile  filaments,  1 5  to  40  /*  long,  which  move 
among  the  blood  cells  as  a  serpent  in  the  grass  (Pasteur). 

In  cultures  it  grows  out  very  slightly  from  the  line  of  stab,  giving  a  jagged 
granular  line,  differing  from  tetanus.  Spores  form  best  at  37°  C. — requiring  about 
forty-eight  hours.  It  liquefies  gelatin.  In  examining  an  exudate  from  a  suspected 
case,  note  the  presence  of  spores  centrally  situated.  Inoculate  a  guinea-pig.  Death 
occurs  in  about  two  days.  There  is  intense  hemorrhagic  emphysematous  oedema  at 
the  site  of  inoculation,  the  cedematous  fluid  however  does  not  show  spores.  The 
bacilli  do  not  appear  in  the  blood  until  about  the  time  of  death  and  it  is  an  assistance 
in  diagnosis  to  put  the  dead  body  of  the  guinea-pig  in  the  incubator  for  a  few  hours. 
The  subcutaneous  tissue  contains  fluid  and  gas.  There  is  present  the  foul  odor  of 
an  anaerobe.  Examine  for  the  long  filaments  showing  flowing  motility.  Be  sure 
to  stain  by  Gram.  (Negative.)  For  cultures,  heat  the  material  (either  from  a 
wound  or  from  a  guinea-pig)  which  shows  spores  to  a  temperature  of  80°  C.  for 
from  fifteen  minutes  to  one  hour.  Then  inoculate  glucose  agar  stab  culture  and  grow 
anaerobically.  Courmont  differentiates  anthrax  from  malignant  oedema  by  in- 
jecting into  ear-vein  of  rabbit.  The  injection  of  malignant  oedema  in  this  way, 
instead  of  subcutaneously,  tends  to  immunize. 

B.  botulinus  (Van  Ermengem,  1896). — This  is  the  organism  which 
produces  botulism,  a  form  of  meat  poisoning.  It  is  a  spore-bearing 
anaerobe  and  must  not  be  confused  with  another  organism  associated 
with  meat  poisoning — the  B.  enteritidis  of  Gartner.  The  spores  are 
at  the  end  and  are  not  very  resistant;  a  temperature  of  80°  C.  often 
killing  them. 

In  botulism  the  meat  becomes  infected  after  the  animal  has  been  slaughtered; 
in  Gartner  meat  poisoning  the  cow  meat  was  infected  at  the  time  of  slaughter — it 
was  from  a  sick  animal.  Thorough  cooking  of  the  meat  protects  against  botulism 
but  not  certainly  against  Gartner  meat  poisoning. 

There  are  dysphagia,  paralysis  of  eye-muscles,  and  cardiac  and  respiratory 
symptoms  (medulla).  The  symptoms  are  due  to  the  elaboration  of  a  soluble  toxin 
of  the  same  nature  as  that  of  diphtheria  and  tetanus.  There  is  no  fever  and  con- 
sciousness is  preserved. 

An  antitoxin  which  it  is  stated  has  therapeutic  value  in  botulism  has  been 


BOTULISM  71 

prepared  in  the  usual  way  by  Kempner.     Without  serum  treatment  death  occurs  in 
about  40%  of  cases  and  takes  place  between  twenty-four  and  forj^eight  hours. 

The  bacillus  has  been  isolated  from  sausage  and  ham.     It  is  a  large  bacillus — 
5  to  io/iX  ij".     It  is  slightly  motile  and  stains  by  Gram.     It  produces  gas  in  glucose 


Fig.   21. — Bacillus  of  botulism,     (Kolle  and  Wassermann.} 


FIG.    22.— Symptomatic   anthrax    (Rauschbrand)  bacilli    showing  spores.      (Kolle 

and  Wassermann.) 


media.     It  grows  best  at  22°  and  only  slightly  at  37° — hence  it  is  dangerous  only 
irom  its  soluble  toxin,  the  bacilli  not  developing  to  any  extent  in  the  body. 

For  this  reason  botulism  patients  are  not  a  source  of  danger,  it  is  the  infected 
meat  alone  which  causes  the  disease.  On  the  contrary  where  the  meat  poisoning 
is  due  to  the  Gartner  or  paratyphoid  group  infection  may  take  place  from  the  patient's 
discharges. 


72  STUDY   AND    IDENTIFICATION    OF   BACTERIA 

When  the  toxin  is  introduced,  it  requires  a  period  of  incubation  of 
twelve  to  twenty  hours.  Symptoms  of  gastrointestinal  disorder  may 
come  on  shortly  after  the  ingestion  of  the  toxin  containing  food,  these 
however  are  not  the  specific  manifestations,  as  are  the  eye  symp- 
toms, etc. 

An  important  point  is  that  ham  may  not  appear  decomposed  and  yet  contain 
many  bacilli  and  much  toxin.  It  is  a  very  potent  toxin — as  little  as  one-thousandth 
of  a  c.c.  may  kill  a  guinea-p'ig.  In  man  the  toxin  is  apparently  absorbed  from  the 
alimentary  canal.  For  diagnosis  inject  an  infusion  of  the  ham  or  sausage  which 
was  eaten  of  into  a  guinea-pig,  and  characteristic  pupillary  symptoms  with  death 
by  cardiac  and  respiratory  failure  will  result. 

Cultures  may  be  made  in  glucose  agar. 

The  culture  is  disrupted  by  gas.  Incubation  at  room  temperature  and  in  the 
dark  is  necessary.  There  is  a  rancid  odor.  The  characteristic  point  is  the  pro- 
duction of  a  powerful  soluble  toxin  which  produces  symptoms  when  no  bacilli  are 
present. 

B.  tetani  (Nicolaier,  1885;  Kitasato,  1889).— This  is  the  most 
important  organism  of  the  anaerobic  spore  bearers.  Its  characteristics 
are  the  tetanic  symptoms  produced  by  the  toxin  and  the  strictly  termi- 
nal drum-stick  spores.  Spores  are  difficult  to  find  in  material  from 
wounds  infected  with  tetanus,  but  readily  develop  in  cultures.  Prior  to 
the  formation  of  spores  the  organism  is  a  long  thin  bacillus  (4  X  0.4^) .  It 
is  motile  and  Gram  positive.  It  liquefies  gelatin  slowly  and  does  not 
coagulate  milk. 

Theobald  Smith  recommends  growing  it  in  fermentation  tubes  containing 
ordinary  bouillon,  but  to  which  a  piece  of  the  liver  or  spleen  of  a  rabbit  or  guinea-pig 
has  been  introduced  at  the  junction  of  the  closed  arm  and  the  open  bulb.  By  this 
method  spores  develop  rapidly  in  from  twenty-four  to  thirty-six  hours.  Sporulation 
is  most  rapid  at  37°  C.  As  there  is  always  liability  to  postmortem  invasion  of 
viscera  by  ordinary  saprophytes,  Smith  recommends  that  great  care  be  taken  not 
to  handle  the  animal  roughly  in  chloroforming  and  in  pinching  off  pieces  of  the 
organ  at  autopsy.  The  animal  must  be  healthy,  and  the  tubes  to  which  the  piece 
of  tissue  is  added  must  be  proven  sterile  by  incubation.  Smith  calls  attention  to 
the  uncertainty  of  the  temperature  at  which  tetanus  spores  are  killed.  He  shows 
that  some  require  temperature  only  possible  with  an  autoclave.  In  view  of  the 
danger  of  tetanus,  it  is  advisable  to  carefully  autoclave  all  material  going  into  bac- 
terial vaccines,  such  as  salt  solution,  bottles  for  holding,  etc. 

Tetanus  seems  to  grow  better  in  symbiosis  with  aerobes;  hence  a 
lacerated  dirty  wound  with  its  probable  contamination  with  various 
cocci,  etc.,  and  its  difficulty  of  sterilization,  offers  a  favorable  soil.  The 
tetanus  bacillus  gives  rise  to  one  of  the  most  powerful  poisons  known; 


TETANUS  73 

it  is  a  soluble  toxin  like  diphtheria  toxin,  and  it  is  estimated  that  1/300 
of  a  grain  is  fatal  for  man. 

Rosenau  has  established  an  antitoxin  unit  for  tetanus  which  has  the  power  of 
neutralizing  one  thousand  minimal  lethal  doses.  Practically,  it  is  ten  times  the 
least  quantity  of  antitetanic  serum  necessary  to  protect  the  life  of  a  350  grams 
guinea-pig  from  a  test  dose  of  tetanus  toxin  furnished  by  the  hygienic  laboratory. 
(The  necessity  of  some  definite  unit  is  apparent  when  tests  have  shown  that  serum 
stated  to  contain  six  million  units  per  c.c.  only  had  a  value  of  90  of  the  official 
American  units.)  Consequently  it  is  a  unit  ten  times  as  neutralizing  as  the  diph- 
theria antitoxin  one.  The  antitoxin  of  tetanus  is  less  efficient  than  that  of  diphtheria 
for  the  following  reasons: 

i.  There  is  about  three  times  as  great  affinity  in  vitro  between  diphtheria  toxin 
and  antitoxin  as  is  the  case  with  tetanus. 


FIG.  23. — Tetanus  bacilli  showing  end  spores.     (Kolle  and  Wassermann.) 

2.  The  tetanus  toxin  has  greater  affinity  for  nerve  cells  than  for  antitoxin. 

3.  Treatment  with  antitoxin  is  successful  after  symp.toms  of  diphtheria  appear. 
With  tetanus  it  is  almost  hopeless  after  the  disease  shows  itself.     Hence  the  impor- 
tance of  the  early  bacteriological  examination  of  material  from  a  suspicious  wound 
(rusty  nail). 

4.  The  tetanus  toxin  ascends  by  way  of  the  axis  cylinder,  and  the  antitoxin 
being  in  the  circulating  fluids  cannot  reach  it,  whereas  with  diphtheria  both  toxin 
and  antitoxin  are  in  the  circulation.     Diphtheria  also  selects  the  cells  of  paren- 
chymatous  and  lymphatic  organs  which  are  more  tolerant  of  injury  than  the  nerve 
cells.     The  dose  of  tetanus  antitoxin  as  a  prophylactic  is  1500  units;  as  a  curative 
agent  5000  to  20,000  units.     Recent  experience  shows  that  it  should  be  injected 
intravenously  when  symptoms  have  manifested  themselves. 

That  the  disease  is  due  to  toxin  is  shown  not  only  experimentally,  but  also  if 
spores  are  carefully  freed  of  all  toxin  by  washing,  and  then  introduced  they  do  not 
cause  tetanus — the  polymorphonuclears  engulfing  them.  The  importance  of  the 


74  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

presence  of  ordinary  pus  cocci  in  a  tetanus  wound  may  be  that  the  activity  of  the 
leucocytes  in  phagocytizing  them  allows  the  tetanus  bacillus  to  escape  phagocytosis. 

This  would  also  explain  the  importance  of  nec- 
rotic  tissue  in  a  lacerated  wound — the  phagocytes 
taking  this  up  instead  of  tetanus  bacilli.  The 
toxin  is  digested  by  the  alimentary  canal  juices 
and  infection  by  that  atrium  is  improbable. 
The  infection  occurs  especially  through  skin 
wounds,  and  also  from  those  of  mucous  mem- 
brane. While  tetanus  is  like  diphtheria,  a  dis- 
ease in  which  the  bacilli  are  localized  and  do  not 
spread,  yet  recently  Richardson  has  obtained 
tetanus  bacilli  in  pure  culture  from  the  tributary 
lymphatic  glands  of  a  "rusty  nail"  wound  of 
foot.  The  cultures  inoculated  into  root  of  tail 
of  a  white  rat  caused  the  rat's  death  in  forty- 
eight  hours  with  typical  "seal  gait"  attitude  of 
tetanus  in  rats. 

The  usual  period  before  symptoms  occur  is 
fifteen  days.  The  shorter  the  period  of  incuba- 
tion, the  more  probably  fatal  the  disease.  The 
horse  is  the  most  susceptible  animal,  next  the 
guinea-pig,  then  the  mouse.  Fowls  are  practi- 
cally immune. 

In  examining  for  tetanus,  scrape  out 
the  material  from  the  suspected  wound 
with  a  sterile  Volkmann  spoon  and  put 
it  in  a  tube  containing  blood-serum. 
Place  this  in  an  incubator.  We  have 
here  the  principle  of  the  septic  tank — 
the  cocci  and  other  aerobes  grow  lux- 
uriantly and  enable  the  tetanus  bacillus 
to  develop.  From  day  to  day  smell  the 
culture,  and  if  an  odor  similar  to  the 
penetrating,  sour,  foul  smell  of  the  stools 
of  a  man  who  has  been  on  a  debauch  be 
detected,  it  is  suspicious.  The  nonde- 
velopment  of  a  foul  odor  is  against 
FIG.  24.— B.  aerogenes  capsu-  tetanus.  Also  make  smears  from  the 
latus  agar  culture  showing  gas  material  and  examine  for  drum-stick 
formation.  (Williams.)  T,  ,,  f 

spores.      If    these  are   found,   heat    the 

material  to  80°  C.  for  one-half  hour,  to  kill  nonsporing  aerobes  and 
facultative  anaerobes,  and  then  inoculate  a  deep  glucose  agar  tube 


THE    GAS  BACILLUS  75 

and  cultivate  by  Wright's  method.     The  fusiform  lateral  outgrowth 
about  the  middle  of  the  stab  is  characteristic. 

A  more  rapid  method  is  to  draw  up  the  material,  provided  it  be  pus  (tissue 
scrapings  may  be  emulsified  in  sterile  salt  solution)  into  a  capillary  bulb  pipette. 
Then  seal  off  the  end  and  heat  the  capillary  bulb  pipette  and  its  contents  in  a  water 
bath  at  80°  C.  for  15  minutes.  Next  break  off  the  sealed  tip  and  stick  the  pipette 
into  a  deep  tube  of  glucose  agar.  When  the  point  reaches  the  bottom,  force  out  the 
material  along  the  line  of  the  stab  as  the  pipette  is  withdrawn.  Cover  the  surface  of 
the  agar  with  sterile  liquid  petrolatum  and  incubate.  Better  anaerobic  conditions 
obtain  where  the  Buchner  or  Wright  method  is  employed. 

Tetanus  produces  no  gas.  Material  for  examination  is  best  obtained  with  a  bulb 
pipette  (containing  a  little  sterile  salt  solution)  which  is  plunged  into  the  agar  and  the 
salt  solution  forced  out  and  drawn  in  where  a  proper  growth  is  noted. 

Spores  form  in  thirty-six  to  forty-eight  hours.  In  injecting  test  animals  it  is 
advisable  to  divide  the  material  to  be  injected  into  two  portions;  one  animal  is 
injected  with  the  material  alone,  the  second  animal  with  tetanus  antitoxin  at  the 
same  time  the  material  is  injected.  Only  the  first  animal  dies  with  tetanic  symptoms. 

B.  aerogenes  capsulatus  (Welch,  1891).— This  bacillus  is  appar- 
ently widely  distributed.  It  is  possibly  the  same  organism  as  Klein's 
B.  enteritidis  sporogenes,  which  is  constantly  present  in  faeces.  It  is  a 
large  capsulated  organism,  which  does  not  form  chains.  Spores  are 
produced  on  blood-serum.  These  are  frequently  absent  on  other 
media.  It  is  questioned  whether  its  pathogenicity  is  other  than  ex- 
ceedingly feeble,  the  presence  of  the  bacillus  in  emphysematous  find- 
ings at  postmortem  being  attributed  to  terminal  or  cadaveric  invasion. 

Cases,  however,  in  the  Philippines,  have  been  reported  following  carabao  horn 
wounds,  in  which  most  serious  and  fatal  results  attended  emphysematous  lesions 
showing  this  bacillus.  The  isolation  of  a  Gram  positive  bacillus  from  a  lacerated 
wound  discharge,  even  in  the  absence  of  emphysema,  is  almost  diagnostic. 

In  milk  cultures  we  have  coagulation  and  from  the  subsequent  development 
of  gas  the  disruption  of  the  coagulum  into  shreds.  An  odor  of  butyric  acid  is 
developed. 

Cultures  in  litmus  milk  show  these  shreds  plastered  against  the  sides  of  the  tube 
and  showing  a  pink  color. 

It  is  the  cause  of  "foamy  organs"  occasionally  present  at  autopsy. 

The  best  method  of  diagnosis  is  to  inoculate  the  culture  or  material 
into  the  ear  vein  of  a  rabbit,  kill  it  and  then  incubate  the  body  at  37°  C. 
Gas  is  generated  in  the  organs  in  a  few  hours. 

Achalme  isolated  a  large  bacillus  from  a  fatal  case  of  rheumatism  which  is  now 
considered  as  having  no  relation  to  acute  rheumatism  and  which  was  probably  B. 
aerogenes  capsulatus. 

Kendall  has  called  attention  to  the  importance  of  this  organism  in  a  certain 
proportion  of  cases  of  summer  diarrhcea  of  infants.  (See  under  chapter  on  faeces.) 


CHAPTER  VII. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.    MYCO- 

BACTERIA  AND  CORYNEBACTERIA.    KEY 

AND  NOTES. 

Key  for  Bacilli. — Having  branching  characteristics,  as  shown  by 
parallelism,  branching,  curving  forms,  V-shapes,  clubbing  at  ends,  seg- 
mental  staining,  etc. 

/  Cultures  more  or  less  wrinkled  and  dry. 
Acid-fast.     Mycobactermm.    \  ,, 

(^  More  like  moulds. 

I.  Grow  rapidly  on  ordinary  media  at  room  temperature. 

Examples:  Timothy  grass  bacillus  of  Moeller  (B.  phlei). 

Mist  bacillus.     Butter  bacilli  as  reported  by  (i)  Rabinowitsch 
and  (2)  Petri. 

II.  Only  grow  at  about  incubator  temperature.     Scanty  growth  or  none  at  all 

on  ordinary  media.     Media  of  preference  are:   (a)   solidified  blood-serum, 
(b)  glycerine  agar,  (c)  glycerine  potato  and  (d)  egg  media. 

1.  Cultures  fairly  moist,  luxuriant,  and  flat.     Op.  temp.  43°  C. 
a.  Bacillus  of  avian  tuberculosis. 

2.  Cultures  scanty,  wrinkled,  and  dry.     Appear  in  ten  to  fourteen  days.     Op. 
temp.  38°  C.     Bacilli  longer,  narrower,  more  regular  in  outline  and  staining 
than  bovine;   vacuolation   more   marked   (2.5^).     Smear  from   organs   of 
inoculated  guinea-pig  shows  few  bacilli.    Less  virulent  for  rabbits. 

a.  Bacillus  of  human  tuberculosis. 

Cultures  as  above,  but  even  more  scanty.     Bacilli  shorter,   thicker,  less 
vacuolated  (1.5^).     Smear  from  organs  of  guinea-pig  shows  many  bacilli. 

b.  Bovine  tubercle  bacilli. 

3.  Very  difficult  to  cultivate  (Czaplewski). 
Smegma  bacilli  of  various  animals. 

III.  Noncultivable  by  ordinary  methods.     Cultivable  in  symbiosis  with  amoebae. 
(Clegg.)     Duval  cultivated  an  acid-fast  bacillus   on  N.N.N.   medium  con- 
taining i%  glycerine.     Bayon  cultivated  on  placental  juice  glycerine  agar  a 
slightly  acid-fast  diphtheroid  which  changed  to  acid  fast  in  peritoneum  of 
mouse.     Bayon's  organism  thought  to  be  similar  to  Kedrowsky's  diphtheroid 
of  leprosy. 

i.  B.  leprae.     Found  chiefly  in  nasal  mucus  and  in  juice  from  lepra  tubercles. 
Less  often  in  nerve  leprosy. 


ACID    FAST  BACILLI  77 

f  Colonies  more  flat  and  moist. 
Nonacid-fast.      Corynebactermm.  <  _.. 

(  Like  other  bacteria. 

I.  Do  not  stain  by  Gram's  method. 

i.  B.  mallei  (Glanders).  Characteristic  culture  is  that  on  potato.  Growth 
like  layer  of  honey  by  third  day.  Becomes  darker  in  color,  until  on  eighth 
day  is  reddish-brown  or  opaque  with  greenish-yellow  margin. 

II.  Gram  positive. 

1.  Very  luxuriant  growth  on  ordinary  media.     Colonies  often  yellow  to  brown- 
ish.    B.  pseudodiphtheriae.     Shorter,  thicker  and  stain  uniformly. 

2.  Moderate   growth   on   ordinary    media.     B.  diphtheriae.     Best  media    are 
blood-serum  (Loffler's)  or  glycerine  agar.     Has  metachromatic  granules  at 
poles. 

3.  Scanty  and  slow  growth  on  nutrient  media.     B.  xerosis. 


THE  GROUP  or  ACID-FAST  BRANCHING  BACILLI. 

There  is  a  large  and  ever-increasing  number  of  organisms  which 
have  the  same  staining  reactions  as  the  tubercle  bacilli,  but  which  differ 
in  four  important  essentials  of: 

1.  Growing  readily  on  any  media. 

2.  Showing  more  or  less  abundant  growth  or  colonies  in  twenty- 
four  hours. 

3.  Having  no  pathogenic  power  for  guinea-pigs  when  inoculated 
subcutaneously. 

4.  Not  requiring  body  temperature  for  development,  but  growing 
at  room  temperature. 

Many  of  these  organisms,  if  injected  intraperitoneally  into  guinea-pigs  will  pro- 
duce a  peritonitis  with  false  membrane.  Some  also  produce  granulation  tissue 
nodules  which  may  be  confused  with  true  tubercles.  For  this  reason  it  is  well  to 
study  the  lesions  in  experimental  tuberculosis  in  the  guinea-pig.  Injected  subcuta- 
neously, on  either  or  both  sides  of  the  posterior  abdomen  with  the  needle  pointing 
toward  the  inguinal  glands,  we  may  have  caseation  and  ulceration  at  the  site  of 
inoculation.  The  glands  in  relation  enlarge  and  caseate.  Smears  from  these  show 
T.  B.  The  marked  and  characteristic  change  is  the  enormous  enlargement  of  the 
spleen,  which  is  studded  with  grayish  and  yellow  tubercles.  Make  smears  and  cul- 
tures from  the  spleen.  The  death  of  the  guinea-pig  usually  occurs  in  about  two 
months.  The  lesions  may  be  looked  for  at  three  to  five  weeks. 

These  nonpathogenic  acid-fast  bacilli  are  of  greatest  importance  by  reason  of 
their  possible  confusion  with  the  true  tubercle  bacilli.  Their  colonies  correspond 
more  or  less  with  different  types  of  tubercle  bacilli  colonies,  being  either  dry  and  wrint 
kled  like  human,  or  moist  and  irregularly  flat  as  avian.  Eventually  the  mois- 
colonies  become  dry  and  wrinkled.  They  have  been  isolated  from: 

1.  Butter  and  milk. 

2.  From  grasses,  especially  in  timothy  grass  infusion. 


78 


STUDY   AND    IDENTIFICATION    OF   BACTERIA 


3.  In  various  excretions  of  animals,  as  in 
dung,  urine,  etc. 

4.  Normally    in    man — from     skin,     nasal 
mucus,  cerumen,  and  tonsillar  exudate. 

It  is  important  to  remember  that  such  organisms 
have  very  rarely  been  reported  from  pulmonary 
lesions,  and  when  present  they  have  been  considered 
as  probably  causative. 

The  present  view  is  that  the  finding  of  tubercle 
bacilli  in  sputum  has  practically  as  great  value  as  it 
had  before  we  knew  of  these  various  acid-fast  bacteria. 

Tubercle  Bacillus  (Koch,  1882). — This  is 
a  rather  long,  narrow  rod,  3X0.3;*.  In  the 
human  type  it  tends  to  show  a  beaded  appear- 
ance, this  not  being  due  to  spores,  however. 
In  the  bovine  type  the  staining  is  more  solid, 
the  organism  shorter  and  thicker,  and  shows 
even  a  more  scanty  growth  than  human  T.  B. 
It  has  been  established  that  many  of  the 
tuberculous  affections  of  man,  especially  those 
of  the  skin,  bone,  and  mesenteric  glands,  are 
of  the  bovine  type,  while,  as  a  rule,  pulmo- 
nary and  laryngeal  lesions  are  of  the  human 
type.  Experiments  by  various  commissions 
in  different  countries  have  shown  that  human 
and  bovine  types  are  very  closely  related  and 
that  not  only  may  a  bovine  strain  affect  man, 
but  that  human  T.  B.  may  infect  young 
calves.  As  bacilli  of  the  bovine  type  have 
frequently  been  reported  in  intestinal  and 
mesenteric  tuberculosis  of  children  it  shows 
the  importance  of  sterilizing  cows'  milk. 
Koch  considers  human  infection  from  bovine 
sources  as  of  very  rare  occurrence. 


FIG.  25. — Bacillus  tuber- 
culosis; glycerine  agar-agar 
culture,  several  months  old. 
(Curtis.) 


Although  Kossel  has  found  only  two  cases  of  bovine 
T.  B.  in  709  cases  of  pulmonary  tuberculosis  yet  for 
the  other  types  the  findings  are  different.  Leaving 
out  of  consideration  the  frequency  of  infections  with 

bovine  T.  B.  in  children,  recent  statistics  have  shown  that  in  adults  about  4%  of 
cervical  adenitis,  22%  of  tabes  mesenterica  and  3.5%  of  bone  and  joint  tuberculosis 
are  due  to  bovine  strains  of  T.  B. 


TUBERCULOSIS  79 

The  British  Royal  Commission  in  its  final  report  of  July,  1911,  considered  three 
types  of  T.  B. 

I.  The  bovine  type  belonging  to  the  natural  tuberculosis  of  cattle. 
II.  The  human  type.     The  type  more  generally  found  in  man. 
III.  The  avian  type,  belonging  to  natural  tuberculosis  of  fowls. 

The  bovine  type  grows  slowly  on  serum  and  at  the  end  of  two  to  three  weeks 
shows  only  a  thin  grayish  uniform  growth  which  is  not  wrinkled  and  not  pigmented. 
The  human  type  grows  more  rapidly  and  tends  to  become  wrinkled  and  pigmented. 
Subcutaneous  inoculation  of  50  mg.  of  culture  into  the  neck  of  calves  produced 
generalized  tuberculosis.  A  similar  injection  of  human  T.  B.  does  not  cause  general- 
ized tuberculosis  but  only  an  encapsulated  local  lesion. 

Intravenous  injection  of  o.oi  to  o.i  mg.  of  bovine  T.  B.  into  rabbits  causes  general 
miliary  tuberculosis  and  death  within  five  weeks.  With  human  T.  B.  in  doses  of 
o.i  to  i.o  mg.,  similarly  injected,  the  majority  of  rabbits  live  for  three  months. 

Subcutaneous  injection  of  10  mg.  bovine  T.  B.  causes  death  in  28  to  101  days. 
Similar  injection  of  human  T.  B.  in  doses  up  to  100  mg.  did  not  kill  the  rabbits  after 
periods  of  from  94  to  725  days.  The  duration  of  life  in  injected  guinea-pigs  is  longer 
with  human  than  with  bovine  T.  B. 

Subcutaneous  injections  of  bovine  T.  B.  into  cats  produces  generalized  tubercu- 
losis while  the  cat  is  resistant  to  human  T.  B.  thus  given. 

Recent  statistics  (Beitzke)  show  tuberculous  lesions  in  58%  of 
adults  at  autopsy — Naegli's  figures  were  about  90%. 

It  is  a  question  whether  the  avian  type  is  absolutely  distinct;  many 
experiments  having  indicated  the  impossibility  of  infecting  fowls 
with  human  T.  B.  Nocard,  by  inserting  collodion  sacs  containing 
bouillon  suspensions  of  human  T.  B.,  claims  to  have  changed  these  to 
the  avian  type.  The  avian  type  grows  at  43°  C.  fairly  luxuriantly ras  a 
moist,  more  or  less  spreading  culture.  It  grows  much  better  on  gly- 
cerinated  agar  than  on  serum.  Morphologically  they  are  like  the 
human  type,  but  show  less  tendency  to  form  compact  masses.  Very 
pleomorphic.  Have  been  reported  from  sputum  of  man  (doubtful). 

Fowls  become  infected  by  intravenous  or  subcutaneous  injection  or  as  the  result 
of  feeding.  After  feeding  the  lesions  are  chiefly  of  the  alimentary  tract;  after  in- 
jections, of  spleen,  liver  and  lungs.  Avian  T.  B.  is  more  virulent  for  rabbits  than 
human  T.  B.  but  less  so  than  bovine  T.  B.  The  mouse  is  the  only  animal  besides  the 
rabbit  in  which  avian  T.  B.  can  cause  a  generalized  tuberculosis.  The  conclusions 
are  that  there  is  no  danger  to  man  from  avian  T.  B.  With  the  bovine  type  it  is 
quite  different  as  nearly  one-half  of  the  deaths  in  young  children  from  abdominal 
tuberculosis  were  due  to  bovine  T.  B.  and  to  that  type  alone.  Not  only  in  children, 
but  in  adolescents  suffering  from  cervical  gland  tuberculosis,  a  large  proportion  were 
caused  by  bovine  types.  The  bovine  type  is  also  an  important  factor  in  lupus. 

There  is  also  a  fish  tuberculosis.     This  organism  grows  much  more 


8o  STUDY   AND    IDENTIFICATION    OF   BACTERIA 

rapidly  than  the  other  types  (three  to  four  days),  and  grows  best  at  24° 
C.,  growth  ceasing  at  36°  C.     The  colonies  are  round  and  moist. 

It  is  certain  that  many  of  the  symptoms  usually  noted  in  the  tuberculous  are  due 
to  secondary  infections.  Pettit,  by  careful  blood  cultures,  obtained  the  pneumo- 
coccus  in  24  cases  and  the  streptococcus  in  36  cases  out  of  130  cases  studied.  He 
used  from  5  to  20  c.c.  of  blood  from  the  vein.  Positive  blood  cultures  were  obtained 
in  68%  of  far-advanced  cases,  45%  of  advanced  cases  and  16%  of  incipient  cases. 

The  best  culture  medium  for  primary  cultures  is  blood-serum  or, 
better,  a  mixture  of  yolk  of  egg  and  glycerine  agar.  Dorset's  egg  me- 
dium is  also  used.  In  subcultures,  either  glycerine  agar,  glycerine 
potato,  or  glycerine  bouillon  make  good  media.  In  inoculating  media 
from  tuberculous  material,  as,  say,  from  a  tuberculous  gland  or,  more 
practically,  from  the  spleen  of  a  guinea-pig,  the  material  must  be 
thoroughly  disintegrated  or  rubbed  on  the  surface  of  the  media  so  that 
individual  bacilli  may  rest  on  the  surface  of  the  culture  media.  In 
growing  in  flasks  in  glycerine  bouillon  a  surface  growth  is  desired.  The 
cylindrical  flask  of  Koch  gives  a  better  support  to  the  pellicle  than  an 
Erlenmeyer  one.  In  inoculating,  a  scale  of  such  a  surface  growth  or  a 
grain  from  the  growth  on  a  slant  should  be  deposited  on  the  surface  of 
the  glycerine  bouillon  in  the  flask. 

Inasmuch  as  the  filtrate  from  cultures  has  little  texic  effect,  the 
poison  is  assumed  to  be  intracellular. 

Koch's  old  tuberculin,  which  was  simply  a  concentrated  glycerine  bouillon 
culture,  is  now  principally  used  in  veterinary  diagnosis.  It  was  prepared  as  follows: 

After  four  to  six  weeks  the  surface  growth  begins  to  sink  to  the  bottom  of  the 
flask.  This  fully  developed  culture  is  evaporated  over  a  water  bath  at  80°  C.  to 
one-tenth  the  original  volume.  It  is  then  filtered,  the  final  product  containing 
about  40%  of  glycerine. 

Koch's  tuberculin  "R"  or  new  tuberculin  was  introduced  in  1897.  In  this, 
virulent  bacilli  are  dried  in  vacuo,  ground  up  in  water  and  centrifuged.  The  first 
supernatant  fluid  (T.  O.)  is  discarded.  Subsequent  trituration  and  centrifugaliza- 
tion,  preserving  each  time  the  supernatant  suspension,  gives  the  new  tuberculin. 
It  has  been  found  at  times  to  contain  virulent  T.  B. 

Koch's  bazillen  emulsion  has  been  more  recently  introduced  by  Koch  (1901). 

This  is  simply  a  suspension  of  ground  up  bacilli  in  20%  glycerine  solution. 
Another  preparation  is  the  bouillon  filtrate  of  Denys.  This  is  the  unheated  filtrate 
of  broth  cultures  of  human  T.  B.  It  contains  1/4%  phenol. 

In  the  use  of  T.  R.  and  of  bazillen  emulsion,  Sir  A.  Wright  recommends  doses 
of  1/4000  of  a  milligram,  and  he  rarely  goes  beyond  i/iooo  of  a  milligram  in  treatment. 
These  products  come  in  i  c.c.  bottles  containing  5  mg.  of  bacillary  material.  It  is 
convenient  to  remove  2/10  of  a  c.c.,  containing  i  mg.  Add  this  to  10  c.c.  of  glycerine 
salt  solution  with  1/4%  of  lysol.  Each  c.c.  contains  i/io  mg.  One  c.c.  of  this  stock 


DIAGNOSIS    OF    TUBERCULOSIS  8 1 

solution  added  to  99  c.c.  of  salt  solution,  with  1/4%  of  lysol,  would  give  a  working 
solution,  each  c.c.  of  which  would  contain  i/iooo  mg.  of  tuberculin. 

For  diagnostic  reactions  we  have,  besides  the  method  of  injecting 
tuberculin  and  noting  presence  or  absence  of  fever,  six  more  recent 
diagnostic  tuberculin  tests:  i.  Variations  in  opsonic  index.  2.  Instil- 
lation into  one  eye  of  a  drop  of  1/2%  or  i%  solution  of  purified  tuber- 
culin. Reaction  is  shown  by  redness,  especially  of  inner  canthus,  in 
twelve  to  twenty-four  hours  (Calmette).  A  previous  instillation  may 
sensitize  a  nontuberculous  case  and  a  second  application  of  the  drop  may 
give  an  erroneous  diagnosis.  3.  The  cutaneous  inoculation  method 
(similar  to  ordinary  vaccination  methods).  Scarify  two  small  areas  on 
the  arm  (i/io  inch  in  diameter),  about  2  inches  apart.  Rub  in  one 
a  drop  of  old  tuberculin,  in  the  other  a  drop  of  25%  tuberculin.  As  a 
control  scarify  a  spot  midway  and  to  one  side  of  the  others  and  rub  in 
one  drop  of  0.5%  carbolic  glycerine.  The  appearance  of  bright  red 
papules  in  twenty-four  hours  indicates  reaction  (von  Pirquet).  This  is 
the  method  of  preference.  4.  Intracutaneous  inoculation  of  one  drop 
of  a  i-iooo,  i-ioo  or  i-io  dilution  of  old  tuberculin  (Mantoux  and 
Moussu).  Webb  recommends  hypodermic  needle  points  which  have 
been  dipped  in  old  tuberculin  and  the  points  allowed  to  dry.  A  drop 
of  water  is  placed  on  the  skin  and  the  needle  points  having  been  mois- 
tened in  it  are  plunged  through  the  skin  and  withdrawn  with  a  twist. 
A  definite  lump  shows  a  positive  reaction.  5.  Ointment  tuberculin 
test.  Rub  in  50%  ointment  of  tuberculin  in  lanolin.  Reaction  is 
shown  by  dermatitis  with  reddened  papules  in  twenty-four  to  forty- 
eight  hours  (Moro).  6.  Inoculation  of  bovine  and  human  tuberculin 
to  diagnose  type  of  infection  (Detre).  Of  questionable  value. 

Ebright  injects  the  suspected  material  into  the  subcutaneous  tissue  of  one  side 
of  the  abdomen  of  three  guinea-pigs.  At  the  end  of  one  week  an  injection  into  the 
other  side  of  the  abdomen  of  one  of  the  guinea-pigs  of  1/4  c.c.  tuberculin  is  given. 
Twenty-four  hours  later  smears  are  made  from  the  original  site  of  inoculation  and 
examined  for  tubercle  bacilli.  If  negative  this  is  repeated  with  a  second  guinea- 
pig  at  the  end  of  the  second  week  and  finally  at  the  end  of  the  third  week  with  the 
third  guinea-pig. 

Bloch's  method  is  to  damage  the  lymphatic  glands  in  the  inguinal  region  by 
squeezing  the  tissue  between  the  fingers.  Injections  made  there  of  tuberculosis 
material  show  abundant  tubercle  bacilli  in  these  damaged  glands  in  ten  to  twelve 
days. 

In  staining  it  is  better  to  use  the  Ziehl-Neelsen  method,  decolorizing 
with  3%  hydrochloric  acid  in  95%  alcohol.     The  alcohol,  for  all  prac- 
6 


82  STUDY   AND    IDENTIFICATION    OF  BACTERIA 

tical  purposes,  enables  us  to  eliminate  the  smegma  and  similar  bacilli, 
these  being  decolorized  by  such  treatment.  There  are  two  objections 
to  the  Gabbett  method,  where  decolorizer  and  counterstain  are  combined : 
i.  We  cannot  judge  of  the  degree  of  decolorization — we  are  working 
in  the  dark;  and  2.  the  matter  of  elimination  of  smegma  bacilli  is 
impossible. 

Pappenheim's  method,  in  which  corallin  and  methylene  blue  are  dissolved  in 
alcohol,  does  not  appear  to  have  an  advantage  over  acid  alcohol.  As  a  practical 
point  when  the  question  of  tuberculosis  of  the  genito-urinary  tract  is  involved, 
inoculate  a  guinea-pig  with  urinary  sediment. 

It  must  be  remembered  that  in  young  cultures  of  tubercle  bacilli  many  of  the 
rods  are  nonacid-fast,  taking  the  blue  of  the  counterstain,  while  older  rods  are  acid- 
fast.  This  frequently  causes  suspicion  of  a  contaminated  culture. 

Discussion  has  arisen  as  to  the  granules  of  Much.  These  are  considered  by  Much 
as  resistant  forms  while  others  consider  them  degeneration  forms  of  tubercle 
bacilli.  At  any  rate  material  containing  only  these  Gram  positive  granules  and  no 
acid-fast  rods  may  when  injected  into  animals  give  rise  to  tuberculosis  and  acid- 
fast  bacilli. 

The  combination  of  the  acid-fast  and  Gram  staining  methods  as  recommended 
by  Fontes  is  very  satisfactory. 

Bacillus  Leprae  (Hansen,  1874). — This  is  the  cause  of  leprosy.  In 
nodular  leprosy  the  organism  is  readily  and  in  the  greatest  abundance 
found  in  the  juice  of  the  tubercles  of  the  skin,  and  secretions  of 
ulcerations  of  nasal  and  pharyngeal  mucosa. 

The  earliest  lesion  is  probably  a  nasal  ulcer  at  the  junction  of  the  bony  and 
cartilaginous  septum.  Scrapings  from  this  ulcer  may  give  an  early  diagnosis. 

In  the  skin  they  are  chiefly  found  in  the  derma  packed  in  the  so-called  lepra 
cells.  The  process  is  granulomatous  but  does  not  show  the  caseation  of  tubercu- 
losis or  the  predominant  plasma  cells  of  syphilis.  The  bacilli  are  also  found  engulfed 
in  the  endothelial  cells  lining  the  lymphatics. 

They  are  also  found  in  the  glands  in  relation  to  the  superficial  lesions.  The 
bacilli  are  found  in  smaller  numbers  in  the  liver  and  spleen.  In  anaesthetic  or  nerve 
leprosy  they  are  found  in  small  numbers  in  the  granuloma  tissue  which  affects  the 
interstitial  connective  tissue  of  the  peripheral  nerves.  Also,  rarely,  in  the  anaesthetic 
spots  of  nerve  leprosy. 

In  morphology  and  staining  reactions  they  are  almost  identical 
with  the  tubercle  bacillus.  The  main  points  of  distinction  are :  i.  The 
fact  of  the  leprosy  bacilli  being  found  in  enormous  numbers,  especially 
in  large  vacuolated  cells  (lepra  cells),  and  lying  in  the  lymph  spaces. 
They  are  frequently  beaded  and  lie  in  masses  which  have  been  likened 
to  a  bundle  of  cigars  tied  together,  so  that  smears  show  the  bacilli  in 


LEPROSY  83 

prodigious  numbers.  It  may  be  necessary  to  examine  for  long  periods 
of  time,  smears  made  .from  tuberculosis  lesions  of  skin  before  rinding  a 
single  organism.  2.  Leprosy  bacilli  have  not  been  cultivated  with 
absolute  certainty  3.  Injected  into  guinea-pigs,  they  do  not  produce 
lesions. 

There  have  been  many  reports  of  positive  findings  with  the  Wassermann  test  in  cases 
of  tubercular  leprosy  but  such  reports  are  considered  doubtful  by  many.  Butler, 
in  the  Philippines,  has  found  that  the  lepers  gave  no  higher  percentage  of  positive 
Wassermann  reactions  than  did  the  nonleprous  native  patients  at  his  clinic. 

Recently  a  leprosy-like  disease  of  rats  has  been  reported  in  which  there  are  two 
types:  i.  A  skin  affection  and  2.  a  glandular  one.  In  this  disease,  acid-fast 
bacilli,  alike  in  all  respects  to  leprosy  bacilli,  have  been  found.  Deanehas  obtained 
a  diphtheroid-like  organism  in  culture,  which  is  nonacid-fast.  This  same  finding 
has  been  obtained  in  cultures  considered  positive  in  human  leprosy. 

Quite  recently  it  has  been  claimed  that  the  leprosy  bacillus  has  been 
cultivated  by  excising  aseptically  the  subcutaneous  portion  of  lepromata 
and  dropping  the  leprous  tissue  into  salt  solution,  the  resulting  growth 
being  like  a  streptothrix.  This  was  the  basis  of  the  Nastin  treatment 
which  is  now  more  or  less  discredited.  In  1909  Clegg  reported  that  by 
smearing  plates  containing  amoebae  with  spleen  pulp  of  lepers  (in  which 
the  bacilli  were  abundant)  he  obtained  growth  of  an  acid-fast  bacillus. 
He  was  able  to  carry  on  these  organisms  in  subculture  for  several  genera- 
tions. A  vaccine  made  from  these  bacilli  does  not  seem  to  have  been 
successful. 

Duval  states  that  he  has  cultivated  the  lepra  bacillus  on  Novy-Mac- 
Neal  media  to  which  i%  glycerine  had  been  added.  He  states  that 
white  mice  can  be  inoculated  and  a  pure  culture  obtained  from  the 
peritoneal  cavity.  According  to  Duval  it  grows  best  at  32°  to  35°  C. 
and  is  not  killed  by  a  temperature  of  60°  C.  It  is  most  easily  obtained 
by  injecting  white  mice  intraperitoneally  with  material  from  leprous 
tissues. 

Bayon  considers  the  cultures  of  Duval  and  Clegg  as  not  shown  to 
have  characteristics  which  would  separate  them  from  the  saprophytic 
group  of  acid-fast  organisms.  He  thinks  that  Kedrowsky's  nonacid- 
fast  diphtheroid  is  one  stage  in  the  typical  acid-fast  leprosy  bacillus. 
He  states  that  sera  of  lepers  showed  the  complement  fixation  test  with 
antigen  made  from  cultures  isolated  by  himself  as  well  as  with  the  Ke- 
drowsky  culture,  which  tests  were  negative  with  Duval's  culture. 

For  diagnosis  we  should  use  both  smears  from  the  nasal  mucus  and 


84  STUDY   AND    IDENTIFICATION    OF   BACTERIA 

from  ulcerated  lepromata  or  from  the  scrapings  from  intact  tubercles. 
Some  advise  centrifuging  with  salt  solution,  but  this  is  rarely  necessary. 
The  most  practical  method  is  by  taking  a  capillary  bulb  pipette  which  has  been 
drawn  out  into  a  fine  point.  The  point  is  broken  off  as  with  a  Wright's  blood  sticker 
and  inserted  deep  into  the  corium.  The  serum  which  results  is  drawn  up,  smeared 
out  on  a  slide  and  stained.  The  best  method  is  to  excise  a  small  portion  of  skin 
or  mucous  membrane,  fix  it  in  absolute  alcohol  or  Zenker's  fluid.  Cut  thin  sections 
in  paraffin.  Stain  with  carbol  fuchsin,  decolorize  with  acid  alcohol,  and  then  stain 
with  haematoxylin.  This  gives  the  location  of  the  bacilli.  This  is  also  a  good 
method  for  tuberculous  tissues.  It  is  claimed  that  the  B.  leprae  stains  more  easily 
and  loses  its  color  more  rapidly  than  the  tubercle  bacillus.  Some  prefer  to  stain 
the  leprosy  bacillus  by  Gram's  method,  it  as  well  as  the  tubercle  bacillus  being 
Gram  positive. 

NONACID-FAST    BRANCHING    BACILLI. 

Bacillus  mallei  (Loffler  and  Shutz,  1882). — This  is  the  cause  of  a 
rather  common  disease  of  horses.  When  affecting  the  superficial 
lymphatic  glands,  it  is  termed  " farcy;"  when  producing  ulceration  of 
nasal  mucous  membrane,  the  term  " glanders"  is  used. 

In  man  there  are  two  types  of  glanders — chronic  and  acute.  In  the 
chronic  form  an  abrasion  becomes  infected  from  contact  with  glanders 
material  and  an  intractable  foul  discharging  ulceration  results.  This 
may  persist  for  months  with  lymphatic  involvement  or  may  become 
acute.  The  acute  form  may  also  develop  from  the  start  and  the  cases 
are  usually  diagnosed  as  pyaemia.  Death  invariably  results  in  acute 
glanders.  The  bacillus  is  a  narrow,  slightly  curved  rod,  about  3X0.3;*. 
It  is  nonmotile  and  Gram  negative.  It  at  times  presents  a  beaded 
appearance.  In  subculture  on  agar  or  blood-serum  the  growth  is 
somewhat  like  typhoid  but  more  translucent.  In  original  cultures  from 
pus  or  tissues  the  colonies  may  not  show  themselves  for  forty-eight 
hours. 

As  the  organism  does  not  tend  to  invade  the  blood  stream,  blood  cultures  are 
apt  to  be  negative.  The  glanders  bacillus  grows  best  on  an  acid  glycerine  agar 

(  +    2). 

The  characteristic  culture  is  that  on  potato.  Grown  at  37°  C.,  we 
have  a  light  brown  mucilaginous  growth,  which  by  the  end  of  a  week 
spreads  out  and  takes  a  cafe  au  lait  color.  The  potato  assumes  a  dirty- 
brown  color.  This  and  the  inoculation  of  a  guinea-pig  are  the  chief 
diagnostic  measures.  If  the  material  is  injected  intraperitoneally  into 
a  male  guinea-pig,  marked  swelling  of  the  testicles  is  noted  within 


DIPHTHERIA  85 

forty-eight  hours,  at  the  earliest,  to  seven  to  ten  days.  Cultures 
should  be  made  from  this  swollen  testicle  as  other  organisms  than 
glanders  may  bring  it  about. 

Only  the  B.  pyocyaneus  and  cholera  vibrios  give  a  similar  coloration  of  potato. 
These  organisms,  however,  are  easily  differentiated.  The  glanders  bacillus  is  the 
most  dangerous  of  laboratory  cultures  and  should  be  handled  with  extreme  care. 

The  best  stains  are  carbol  thionin  and  formol  fuchsin.  In  sections  stained  with 
carbol  thionin  the  bacilli  are  apt  to  be  decolorized  by  the  subsequent  passage  of 
the  section  through  alcohol  and  xylol.  This  may  be  avoided  by  blotting  carefully 
after  the  thionin,  then  clearing  with  xylol  or  some  oil  and  mounting.  Nicolle's 
tannin  method  is  a  good  one. 

Mallein  is  prepared  by  sterilizing  cultures  that  have  grown  in  glycerine  bouillon 
for  about  a  month  by  means  of  heat  (100°  C.).  The  dead  culture  is  then  filtered 
through  a  Berkefeld  filter  and  the  filtrate  constitutes  mallein.  It  is  chiefly  used  as 
a  means  of  diagnosing  the  disease  in  horses.  The  reaction  consists  in  rise  of  tempera- 
ture and  local  oedema.  The  dose  is  about  i  c.c. 

Agglutination  and  complement  fixation  tests  are  also  used  for  diagnosing  glanders. 

Bacillus  diphtherias  (Klebs  discovered,  1883;  LofHer  cultivated, 
1884). — The  diphtheria  bacillus  is  found  not  only  in  the  false  mem- 
brane which  is  so  characteristic  of  the  disease,  but  may  be  found  in 
abundance  in  the  more  or  less  abundant  secretions  of  nose  and  pharynx. 
In  studying  the  epidemiology  of  diphtheria,  especial  attention  must  be 
given  to  the  examination  of  nasal  discharges. 

Infection  of  the  larynx  and  middle  ear  are  not  very  rare.  The 
mucous  membrane  of  the  vagina  or  the  conjunctiva  may  also  be  infected. 
The  B.  diphtherias  may  be  in  pure  culture  lying  entangled  in  the  fibrin 
meshes  or  contained  within  leukocytes  in  the  membrane  or  be  asso- 
ciated with  staphylococci,  pneumococci,  or  especially  streptococci. 
These  latter  complicate  unfavorably  and  cause  the  suppurative  con- 
ditions about  the  neck.  In  fatal  cases  the  diphtheria  bacillus  may  be 
found  in  the  lungs.  Ordinarily,  however,  it  remains  entirely  local  and 
does  not  get  into  the  circulation  or  viscera. 

It  produces  soluble  absorbable  poisons  which  are  designated  toxin 
in  the  case  of  the  one  responsible  for  the  acute  intoxication,  paren- 
chymatous  degeneration  and  death;  and  toxone  for  the  poison  which 
produces  cedema  at  the  site  of  inoculation  and  postdiphtheritic  palsy. 
The  injection  of  the  soluble  poisons  alone  without  the  bacilli  produces 
the  symptoms  of  the  disease. 

The  bacilli  tend  to  appear  as  slightly  curved  rods,  showing  varying 
irregularities  in  staining,  as  banding  or  beading,  and  in  particular  the 


86  STUDY   AND    IDENTIFICATION   OF   BACTERIA 

presence  at  either  end  of  small,  deeply  staining  dots  (metachromatic 
granules).  These  granules  may  be  seen  in  an  eighteen-hour  culture, 
but  within  thirty-six  hours,  may  be  abundant. 

These  are  well  seen  with  Loffler's  blue,  but  better  with  Neisser's  method.  In 
culture  they  also  show  swelling  at  one  or  both  ends  or  clubbing.  In  secretions  or  in 
culture  they  show  V-shapes  or  false  branching  and,  what  is  most  characteristic, 
the  parallelism — four  or  five  bacilli  lying  side  by  side  like  palisades.  Being  a  Gram 


FIG.  26. — Bacillus  of  diphtheria.     (X  1000.)     (Williams.) 

positive  organism  while  the  majority  of  the  other  pathogenic  bacilli  are  Gram 
negative,  it  is  of  greatest  importance  to  stain  smears  by  this  method.  It  is  not  so 
strongly  tenacious  of  the  gentian  violet  as  the  cocci,  so  decolorization  should  not  be 
carried  too  far. 

The  best  medium  for  growing  it  is  Loffler's  blood-serum. 

An  egg  medium,  made  of  the  whole  egg  with  glucose  bouillon  as  described  pre- 
viously, is  as  suitable  as  Loffler's  serum.  Coagulated  white  of  egg  answers  fairly 
well,  as  will  a  hard-boiled  egg — the  shell  at  one  end  being  cracked  and  the  white  cut 
with  a  sterile  knife.  This  smooth  side  is  then  inoculated  and  the  egg  placed  cut 
side  downward  in  a  sherry  glass.  If  an  incubator  is  not  at  hand  a  tube  may  be  carried 
next  the  body  in  a  pocket.  The  bacillus  grows  better  on  glycerine  agar  than  on 
plain  agar.  On  such  plates  they  appear  as  small,  coarsely  granular  colonies  with  a 
central  dark  area.  In  size  the  colonies  resemble  the  streptococcus.  On  blood- 
serum  the  colonies  are  larger — 1/12  to  1/8  inch  in  diameter. 

The  diphtheria  bacillus  grows  luxuriantly  on  blood  agar  and  like  the  streptococcus 
pyogenes  has  a  yellowish  laked  zone  around  the  colony.  The  Hofman  and  the 
Xerosis  bacillus  do  not  seem  to  have  this  haemolytic  power.  In  bouillon  it  tends 


DIPHTHERIA   ANTITOXIN  87 

to  form  a  surface  growth.  It  is  at  the  surface  that  the  toxin  function  is  most  marked, 
hence  in  growing  diphtheria  for  toxin  formation  we  use  Fernbach  flasks  which  expose 
a  large  surface  to  the  air.  It  is  a  marked  acid  producer — bouillon  with  a  + 1  reaction 
becoming  +2.5  to  +3  in  thirty-six  hours.  The  filtrate  from  a  two-  or  three- weeks- 
old  broth  culture  is  highly  toxic,  and  is  usually  referred  to  as  diphtheria  toxin.  It 
is  used  in  injecting  horses  to  produce  antitoxin.  Ehrlich  uses  as  a  standard  to  meas- 
ure the  toxicity  of  toxin  the  minimal  lethal  dose  (M.  L.  D.).  This  is  the  amount 
of  toxin  which  will  kill  a  35o-gram  guinea-pig  in  just  four  days.  Some  toxins  have 
been  produced  whose  M.  L.  D.  was  1/500  c.c.,  so  that  i  c.c.  of  such  toxin  would  kill 
500  guinea-pigs.  Theoretically,  the  measure  of  an  antitoxin  unit  is  the  capacity 
of  neutralizing  200  units  of  a  pure  toxin.  (On  exposure  to  light,  etc.,  toxin  loses 


FIG.  27. — Diphtheria  bacilli  involution  forms.     (Kolle  and  Wassermann.) 

its  toxic  power  and  is  termed  toxoid.)  Inasmuch,  however,  as  toxoneand  toxoid 
are  also  present,  we  may  practically  consider  an  antitoxin,  or  immunizing  unit 
(i.e.,  Immunitatseinheit)  as  about  capable  of  making  innocuous  100  M.  L.  D. 

In  the  preparation  of  antitoxin  horses  are  employed;  the  method  being  to  inject 
the  bouillon  filtrate  or  toxin  subcutaneously  at  weekly  intervals  for  a  period  of  three 
or  four  months.  When  each  c.c.  of  the  serum  of  the  horse  is  found  to  contain  about 
250  to  500  antitoxin  units  the  horse  is  bled  from  the  jugular  vein.  Some  sera  contain 
as  much  as  1300  units  in  a  cubic  centimeter. 

Methods  of  purifying  and  concentrating  antitoxin  are  now  employed  by  certain 
makers,  the  principle  being  that  the  antitoxin  in  the  horse  serum  is  precipitated 
with  the  globulins  which  come  down  on  half  saturation  with  ammonium  suplhate. 
In  this  way,  as  the  content  in  horse  serum  proteids  is  lessened,  the  anaphylactic 
dangers  are  lessened. 

As  a  curative  measure,  from  2500  to  5000  units  should  be  injected. 
If  the  injection  is  delayed  or  the  case  very  serious  the  dose  should  be 
10,000  units.  As  much  as  50,000  units  has  been  given  in  severe  cases. 
The  prophylactic  dose  is  500  units. 


88  STUDY    AND    IDENTIFICATION    OF   BACTERIA 

Sudden  death  after  administration  of  antitoxin  has  been  reported 
in  cases  of  status  lymphaticus.  (See  anaphylaxis). 

In  obtaining  material  from  a  throat,  be  sure  that  an  antiseptic  gargle 
has  not  been  used  just  prior  to  taking  the  throat  swab.  The  part  of  the 
swab  which  touched  the  membrane  or  suspicious  spot  should  come  in 
contact  with  the  serum  slant.  This  is  best  accomplished  by  revolving 
the  swab.  An  immediate  diagnosis  is  possible  in  probably  35%  of  cases 
by  making  a  smear  from  a  piece  of  membrane.  In  doing  this  Neisser's 
stain  or  the  toluidin  blue  stain  are  usually  considered  the  most  satis- 
factory. I  prefer  the  Gram  stain,  however.  The  diphtheria  bacilli 
found  in  such  smears  are  not  apt  to  be  clubbed  and  stain  more 
uniformly. 

If  there  is  any  doubt  about  the  nature  of  an  organism  in  a  throat  culture,  always 
stain:  i.  with  Loffler's  alkaline  methylene  blue  for  two  minutes;  2.  with  Gram's 
method,  being  careful  not  to  carry  the  decolorization  too  far,  and  3.  by  Neisser's 
method.  With  Loffler's  you  obtain  a  picture  which,  after  a  little  experience,  is 


FIG.  28. — B.  diphtherias  stained  by  Neisser's  method.     (Williams.') 

characteristic;  at  times  the  polar  bodies  show  as  intense  blue  spots  in  the  lighter 
blue  bacillus.  One  is  liable  to  confuse  cocci  lying  side  by  side  for  diphtheria  bacilli 
with  segmental  or  banded  staining.  This  difficulty  is  not  apparent  when  Gram's 
staining  is  used.  This  gives  us  great  information,  as  the  diphtheria  and  the  pseudo- 
diphtheria  are  the  only  small  Gram  positive  bacilli  usually  found  in  the  mouth. 
The  cocci  are  also  well  brought  out.  Neisser's  stain  gives  a  picture  which,  when 
satisfactory,  is  almost  absolutely  characteristic.  You  have  the  bright  blue  dots 
lying  at  either  end  of  the  light  brownish-yellow  rods.  When  first  isolated  from  a 
throat,  the  diphtheria  bacillus  is  apt  to  stain  characteristically  by  Neisser.  Later 


DIPHTHEROID   BACILLI  89 

on,  in  subculture,  there  may  be  no  staining  of  the  polar  bodies.  Neisscr  originally 
recommended  five  seconds'  application,  with  an  intermediate  washing,  for  each  of 
his  two  solutions.  Thirty  seconds  for  each  is  probably  preferable.  Some  authorities 
recommend  five  to  thirty  minutes.  It  is  well  to  bear  in  mind  that  about  2%  of  the 
people  in  apparent  health  carry  diphtheria  bacilli  of  the  granular  or  barred  type  in 
their  throats  and  of  these  about  one  in  five  will  prove  virulent  for  the  guinea-pig. 

It  is  essential  when  a  question  exists  as  to  the  nature  of  a  diphtheria- 
like  organism  to  test  it  as  to  virulence.  While  there  are  exceptions, 
especially  in  freshly  isolated  colonies,  yet  as  a  rule  a  severe  infection 
yields  virulent  organisms  and  vice  versa.  Pure  cultures  are  best  obtained 
by  streaking  material  from  the  throat  on  glycerine  agar  plates.  From 
an  isolated  colony  inoculate  a  tube  of  bouillon.  From  such  a  twenty- 
four-hour-old  culture  inoculate  a  guinea-pig  with  two  or  three  drops 
subcutaneously  in  the  shaven  abdomen.  Escherich  considers  a  fatal 
result  with  1.5  c.c.  of  such  a  bouillon  culture  a  satisfactory  test  as  to 
virulence.  After  death,  which  occurs  in  two  or  three  days,  the  adrenals 
are  enlarged  and  haemorrhagic. 

Diphtheroid  Bacilli.  Pseudodiphtheria  Bacillus.  Hofman's  Bacil- 
lus.— Under  these  terms  various  Gram  positive  bacilli  have  been  de- 
scribed as  occurring  in  nose  and  in  skin  diseases. 

Their  chief  importance  is  in  connection  with  their  presence  in  the 
throats  of  healthy  people.  Probably  approximately  10%  of  people 
harbor  such  organisms  as  against  i  to  2%  with  granular  types.  Some 
authorities  believe  it  possible  for  these  diphtheroids  to  be  capable  of 
being  transformed  into  virulent  diphtheria  bacilli.  This  seems  im- 
probable. Such  organisms  are  often  found  in  urethral  discharges, 
either  alone,  or  with  gonococci  or  other  organisms. 

1.  They  very  rarely  give  the  blue  dot  staining  at  the  two  ends.     Exceptionally 
they  may  give  a  dot  at  one  end.     Neisser  attaches  importance  to  the  dots 
at  both  ends  as  showing  diphtheria. 

2.  They  tend  to  stain  solidly  or  at  most  with  only  a  single  unstained  segment. 
They  are  shorter,  thicker,  and  do  not  curve  so  gracefully  as  the  true  diphtheria 
bacillus.     They  are  stockier. 

3.  They  produce  very  little  acid  in  sugar  media,  not  one-half  that  produced  by 
true  diphtheria. 

4.  They  are  nonpathogenic  for  guinea-pigs. 

5.  Many  of  them  grow  quite  luxuriantly  and  often  show  chromogenic  power. 

Xerosis  Bacillus. — This  organism  is  frequently  found  in  normal 
conjunctival  discharges.  There  is  question  as  to  its  pathogenesis,  and 
the  finding  of  this  organism  should  not  exclude  the  previous  presence  of 


QO  STUDY   AND    IDENTIFICATION    OF  BACTERIA 

strictly  pathogenic  organisms,  such  as  the  gonococcus  or  the  Koch- 
Weeks.  It  resembles  the  diphtheria  bacillus  in  being  Gram  positive 
and  showing  parallelism,  but  differs  i.  in  being  nonvirulent  for  guinea- 
pigs;  2.  in  requiring  about  two  days  for  the  appearance  of  colonies;  3. 
in  not  showing  Neisser's  granule  staining,  and  4.  in  producing  very  little 
acid  in  sugar  media. 


CHAPTER  VIII. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.    GRAM 
NEGATIVE  BACILLI.    KEY  AND  NOTES. 

KEY  to  the  recognition  of  nonspore-bearing,  nonchromogenic,  non- 
Gram-staining,  nonbranching  bacilli. 

(NOTE. — Some  books  say  that  the  proteus  group  is  Gram  positive.     It  is,  how- 
ever, usually  negative.) 

Do  not  grow  on  ordinary  media.     Require  blood  agar  (haemophilia  bacteria),  serum 
agar,  or  blood-serum. 

Minute  dew-drop  colonies. 

1.  Influenza  bacillus.     Requires  blood  media. 

2.  Koch- Weeks  bacillus  (conjunctivitis).     Serum  agar  best  medium. 

3.  Miiller's  bacillus  of  trachoma.     Like  Koch- Weeks  bacillus,  but  easier  to 
cultivate. 

4.  Morax  diplobacillus  of  conjunctivitis.     Grows  well  and  produces  little  pits 
of  liquefaction  in  Loffler's  blood-serum. 

5.  Bordet-Gengou  bacillus  of  whooping-cough.     Does  not  grow  on  Loffler's 
serum.     Requires  blood  or  ascitic  fluid  agar. 

6.  Ducrey's   bacillus   (soft   chancre).     Requires   almost  pure  blood.     Forms 
chains. 

Grow  well  on  ordinary  media. 

I.  Cultures  in  litmus  milk.     PINK. 

A.  Nonmotile. 

Lactis  aerogenes  group.     B.  lactis  aerogenes. 

Produce  gas  in  glucose,  lactose,  and  saccharose.  No  liquefaction  of  gelatin. 
Short,  stubby  bacteria,  often  showing  capsules.  Intermediate  between 
the  colon  and  Friedlander  group. 

B.  Motile. 

1.  No nliquef action  of  gelatin. 

a.  B.  coli  group.  Coagulation  of  milk.  No  subsequent  peptonization. 
Gas  in  glucose  and  lactose,  none  in  saccharose.  Indol  produced. 
Neutral  red  reduced. 

2.  Liquefaction  of  gelatin. 

a.  B.  cloacae  group.  Gas  in  glucose,  slight  in  lactose.  Slow  coagulation 
of  milk.  Subsequent  peptonization. 

II.  Cultures  in  litmus  milk.     LILAC. 
A.  Nonmotile  bacilli. 

i.  No  gas  generated  in  glucose  or  lactose  bouillon. 

a.  Haemorrhagic  septicaemia  group.     These  are  oval  bacilli  with  tendency 
to  bipolar  staining. 

91 


g  2  STUDY   AND    IDENTIFICATION    OF   BACTERIA 

Colonies  smaller  and  less  opaque  than  those  of  B.  coli. 

Examples:     B.  pestis,  B.  suisepticus,  B.  cholerae  gallinarum  (chicken 

cholera). 

B.  pseudo tuberculosis  rodentium  (very  similar  to  plague). 

B.  pestis  is  absolutely  nonmotile,    does  not  liquefy  gelatin,  does  not 

produce  indol,  produces  slight  acid  in  glucose  but  not  in  lactose  bouillon, 
b.  Dysentery  group.     Colonies  similar  to  those  of  B.  coli. 

Divided  into  two  classes  according  as  mannite  is  acted  on: 

Those  not  giving  acid — nonacid  group — (Shiga-Kruse). 

Those  giving  acid — acid  group — ( Fl ex ner- Strong). 
2.  Gas  generated  in  glucose  bouillon  not  in  lactose. 

a.  Friedlander  group.     Give  very  viscid,  porcelain-like  colonies. 

Tendency  to  capsule  formation  in  favorable  media. 

Examples:    B.  pneumonise,  B.  capsulatus  mucosus,  B  rhinoscleromatis. 
B.  Motile  bacilli. 

1.  Do  not  liquefy  gelatin. 

a.  Do  not  produce  gas  in  either  glucose  or  lactose  bouillon. 

Typhoid,  or  Eberth  group.  No  indol.  No  coagulation  of  milk.  No 
reduction  of  neutral  red. 

b.  Gas  generated  in  glucose,  not  in  lactose  media.     Milk  not  coagulated. 
Neutral  red  reduced. 

Gartner  group.     This  includes: 

Pathogenic  types  for  man;  as  B.  enteritidis,  B.  icteroides,  B.  paratyphoid 
B,  B.  psittacosis.  Nonpathogenic  for  man;  as  B.  cholerae  suum  (hog 
cholera) . 

2.  Liquefy  gelatin. 

a.  Proteus   group.     Colonies   at   first   round  later   amoeboid,   spreading. 
Produce  gas  in  glucose,  not  in  lactose.     Produces  foul  odor. 
B.  zopfii  type  of  Proteus  group  does  not  liquefy  gelatin;  colonies  at  first 
round,  later  amoeboid,  spreading.     Foul  odor  in  cultures.     Gelatin  stab 
shows  lateral  branching. 

NOTE. — The  Friedlander  and  the  lactis  aerogenes  group,  differing  culturally 
chiefly  in  carbohydrate  fermentation  activities,  organisms  considered  as  belonging 
to  the  Friedlander  group  rather  than  to  the  lactis  aerogenes  group  may  show  acid 
in  litmus  milk.  Where  an  organism  having  the  characteristics  of  B.  coli,  but 
fermenting  saccharose,  is  found,  it  is  termed  B.  coli  communior.  A  non-gas 
producing  colon  type  organism  has  been  designated  B.  coli  anaerogenes.  Cer- 
tain organisrns  which  turn  litmus  milk  lilac  and  which  liquefy  gelatin,  but  do  not 
produce  gas  in  sugar  media,  belong  to  the  "Booker"  group.  Other  organisms 
which  acidify  and  coagulate  litmus  milk  but  do  not  liquefy  gelatin  or  produce  gas 
in  glucose  or  lactose  media  have  been  placed  in  the  "Bienstock"  group.  The 
proteus  or  Hauser  group  is  composed  of  organisms  showing  various  functions; 
Proteus  vulgaris  liquefying  gelatin  rapidly,  P.  mirabilis  slowly  and  P.  zenkeri 
not  at  all. 

GRAM  NEGATIVE  BACILLI  REQUIRING  SPECIAL  MEDIA. 

Bacillus  influenzae  (Pfeiffer,  1892). — This  organism  is  the  type  of 
the  so-called  haemophilic  bacteria— organisms  whose  growth  is  restricted 


INFLUENZA  93 

to  media  containing  haemoglobin.  •  The  influenza  bacillus  seems  to 
grow  better  on  slants  freshly  streaked  with  blood  than  on  those  which 
have  been  made  for  some  time,  and  they  appear  to  grow  better  on  this 
surface  smear  of  blood  than  on  a  mixture  of  agar  and  blood. 

The  influenza  bacilli  are  most  likely  to  be  isolated  from  the  sputum  of  broncho- 
pneumonia  due  to  this  organism.  It  has  also  frequently  been  found  in  the  nasal 
secretions  of  influenza  patients.  Exceptionally,  it  is  present  in  the  blood,  and  has 
been  isolated  in  cases  of  meningitis  from  cerebrospinal  fluid.  It  also  occurs  at  times 
in  anginas,  but  then  usually  associated  with  other  organisms.  Infection  probably 
only  takes  place  by  contact.  It  is  a  very  small  bacillus  which  in  sputum  tends  to 
show  itself  in  aggregations,  especially  centering  about  M.  tetragenus.  It  stains 
rather  faintly  when  compared  with  cocci,  so  that  a  smear  of  sputum  stained  with 
formol  fuchsin  shows  a  deep  violet  staining  for  the  M.  tetragenus  or  other  cocci, 
and  scattered  around  in  a  clump-like  aggregation  we  see  these  minute,  rather  faintly 
stained  rods.  They  also  tend  to  stain  more  deeply  at  either  end,  so  that  they  some- 
times appear  as  diplococci.  Gram's  method,  counterstaining  with  formol  fuchsin. 
is  excellent  for  their  demonstration.  The  red  bacilli  and  the  violet-black  cocci  are 
easily  distinguished. 

To  cultivate  them,  rub  the  sputum,  or  at  autopsy  the  material  from 
a  lung,  on  a  slant  smeared  with  human  blood  (pigeon's  blood  is  also 
satisfactory),  and  then  without  sterilizing  the  loop,  inoculate  a  second 
blood  slant;  then  a  third,  and  possibly  a  fourth.  The  colonies  appear  as 
very  minute  dewdrop-like  points  which  seem  to  run  into  each  other  in  a 
wave-like  way.  To  test  such  colonies  we  should  transfer  a  single  colony 
to  plain  agar  and  blood-serum,  trying  not  to  carry  over  any  blood.  If 
the  least  trace  of  blood  is  carried  over,  they  may  grow  on  agar  or  blood- 
serum.  Organisms  resembling  the  influenza  bacillus  have  been  isolated 
from  whooping-cough.  Such  organisms  have  also  been  found  in  the 
fauces  of  well  persons.  In  many  epidemics  of  influenza  the  bacillus 
has  not  been  isolated,  or  success  has  obtained  in  only  a  small  proportion 
of  the  cases.  Etiological  factors  in  conditions  more  or  less  resembling 
influenza  may  be  the  Streptococcus,  Pneumococcus,  or  M.  catarrhalis. 
The  influenza  bacillus  seems  to  grow  best  in  symbiosis  with  some  other 
organism,  especially  with  S.  pyogenes  aureus. 

Koch -Weeks  Bacillus  (Koch,  1883). — This  produces  a  severe 
conjunctivitis.  It  is  very  common  in  Egypt  and  is  also  a  frequent  cause 
of  conjunctivitis  in  the  Philippines  and  in  temperate  climates. 

Smears  made  from  conjunctival  secretion  show  large  numbers  of 
small  Gram-negative  bacilli,  especially  contained  within  pus  cells,  but 
also  lying  free.  They  are  more  difficult  to  cultivate  than  the  influenza 
bacillus,  but  the  same  general  methods  hold.  The  vitality  of  this 


94  STUDY  AND   IDENTIFICATION   OF  BACTERIA 

organism  is  very  slight  so  that. almost  immediate  transference,  of 
material  is  necessary.  Flies  are  an  important  factor  in  Egypt. 
The  period  of  incubation  is  short,  twelve  to  thirty-six  hours.  The 
best  medium  is  a  mixture  of  glycerine  agar  and  hydrocele  or  ascites 
fluid.  At  first  we  rarely  obtain  pure  cultures.  The  colonies  are 
dewdrop-like  and  first  show  themselves  in  about  thirty-six  hours  in 
incubator  cultures. 


FIG.  29.— The  Koch- Weeks  Bacillus.     (Hansell  and  Sweet.) 

Diplobacillus  of  Morax. — This  organism  causes  mild  blepharo-con- 
junctivitis  chiefly  at  the  internal  angle  of  the  eye.  They  are  about  i  or 
2/J.  long  and  tend  to  occur  in  pairs  or  short  chains.  Some  claim  that 
they  are  Gram  positive. 

Culturally  the  formation  of  little  pits  of  liquefaction  in  Loffler's  serum  within 
twenty-four  hours  which  later  become  confluent  may  be  regarded  as  fairly  character- 
istic. They  do  not  grow  on  nutrient  agar. 

After  two  or  three  days  on  blood-serum  rather  marked  involution 
forms  occur.  While  usually  causing  a  more  or  less  chronic  conjuncti- 
vitis they  may  at  times  produce  a  keratitis. 

Bacillus  of  Chancroid  (Ducrey,  1889). — These  are  short  cocco- 
bacilli,  occurring  chiefly  in  chains.  They  show  bipolar  staining.  They 
grow  best  in  a  mixture  of  blood  and  bouillon. 

Bacillus  of  Bordet-Gengou. — This  bacillus  was  reported  as  the  cause 
of  whooping-cough  by  Bordet  and  Gengou  in  1906.  (Czaplewski  and 
Reyher  had  previously  reported  oval  bipolar  staining  organisms,  as  the 
cause  of  pertussis,  and  other  authors  influenza-like  organisms.) 


PLAGUE  95 

The  bacillus  is  oval,  Gram  negative,  shows  bipolar  staining,  somewhat  resembles 
B.  influenzae  and  grows  only  on  uncoagulated  serum  media,  as  blood  or  ascites  agar. 
The  original  cultures  are  very  scanty  so  that  the  colonies  are  difficult  to  recognize. 
In  subcultures  the  growth  is  more  flourishing.  The  organism  is  only  found  in  white, 
thick,  leukocyte  abounding  sputum,  of  the  beginning  of  the  disease.  Hence  per- 
tussis is  probably  contagious  only  at  the  onset. 

Complement  binding  and  agglutination  reactions  have  been  obtained.  For 
diagnosis  stain  the  sputum.  Remember  that  pertussis  gives  a  mononuclear  leuko- 
cytosis  of  15  to  50  thousand. 

GRAM  NEGATIVE  BACILLI  GROWING  ON  ORDINARY  MEDIA. 

Bacillus  pneumonias  (Friedlander,  1882). — This  organism  is 
responsible  for  about  5%  of  the  cases  of  pneumonia.  It  is  usually 
termed  the  pneumobacillus  to  distinguish  it  from  the  pneumococcus; 
at  other  times  Friedlander's  bacillus.  The  name  of  Fraenkel  attaches 
to  the  pneumococcus.  Morphologically,  it  is  a  short,  thick  bacillus, 
and  in  pathological  material,  as  sputum,  shows  a  wide  capsule.  It  is 
nonmotile  and  Gram  negative.  The  colonies  on  agar  are  of  a  pearly 
whiteness  and  are  markedly  viscid.  On  potato  it  shows  a  thick  viscid 
growth  containing  gas  bubbles.  The  characteristic  culture  is  the  nail 
culture  of  a  gelatin  stab.  The  growth  at  the  surface  is  heaped  up  like  a 
round-headed  nail,  the  line  of  puncture  resembling  the  shaft  of  the  nail. 
It  does  not  liquefy  gelatin.  It  does  not  produce  indol,  and  does  not 
produce  gas  in  lactose  bouillon — differences  from  the  colon  bacillus — 
with  which  it  may  be  confused  in  cultures,  as  it  does  not  then  possess  a 
capsule.  If  in  doubt,  inject  a  mouse  at  the  root  of  the  tail.  Death 
from  septicaemia  occurs  in  two  days.  The  peritoneum  is  sticky  and 
numerous  capsulated  bacilli  are  present  in  the  blood  and  organs.  The 
organisms  which  have  been  isolated  from  rhinoscleroma  and  ozcena 
are  practically  identical  with  the  B.  pneumonias.  This  group  of  organ- 
isms is  generally  referred  to  as  the  Friedlander  group.  Similar  organ- 
isms have  been  isolated  from  the  discharges  of  middle-ear  diseases  and 
in  anginas.  Cases  have  been  reported  where  the  B.  pneumonias  was 
the  cause  of  septicaemia  in  man. 

Bacillus  pestis  (Kitasato,  Yersin,  1894). — This  is  the  organism  of 
plague.  It  is  primarily  a  disease  of  rats.  It  is  the  member  of  the  group 
of  haemorrhagic  septicaemias  (Pasteurelloses),  from  which  man  suffers. 

Other  Pasteurelloses  are  chicken  cholera,  swine  plague,  mouse  septicaemia  and 
rabbit  septicaemia.  This  is  a  widely  distributed  group  and  may  include  saprophytic 
organisms  as  well  as  those  noted  for  their  virulence. 


g  STUDY   AND    IDENTIFICATION    OF   BACTERIA 

B.  cholerae  gallinarum  and  B.  suisepticus  are  approximately  similar  in  size  and 
cultural  requirements  to  B.  pestis.  The  oval  bacillus  with  bipolar  staining  in  smears 
from  tissues  is  very  characteristic  for  both  of  them.  Another  name  for  swine  plague 
(B.  suisepticus)  is  infectious  pneumonia  of  swine.  The  organism  is  chiefly  found  in 
the  lungs. 

Where  the  plague  bacilli  are  found  chiefly  in  the  glands,  we  have 
bubonic  plague;  when  in  lungs,  pneumonic  plague;  when  localized  in 
the  skin  and  subcutaneous  tissue,  the  cellulo-cutaneous;  and  when  as  a 
septicaemia,  septicaemic  plague.  An  intestinal  type  is  recognized  by 


FIG.  30. — Colonies  of  plague  bacilli  forty-eight  hours  old.     (Kolle  and  Wassermann.) 

some  authors.  It  must  be  remembered  that  in  all  forms  of  plague  the 
lymphatic  glands  show  hemorrhagic  oedema;  it  is  in  bubonic  plague, 
however,  that  the  areas  of  necrosis  with  periglandular  oedema  are 
prominent.  Where  the  symptoms  are  slight,  mainly  buboes,  the  term 
pestis  minor  is  sometimes  used;  the  typical  disease  being  termed  pestis 
major.  In  pneumonic  plague  we  have  a  bronchopneumonia. 

In  smears  from  material  from  buboes,  from  sputum,  or  in  blood  smears,  as  well 
as  from  blood  or  spleen  smears  from  experimental  animals,  we  obtain  the  typical 
morphology  of  a  coccobacillus  (1.5X0.5^)  with  very  characteristic  bipolar  staining; 
there  being  an  intermediate,  unstained  area.  Very  characteristic  also  is  the  appear- 
ance in  these  smears  of  degenerate  types  which  stain  feebly  and  show  coccoid  and 
inflated  oval  types.  The  presence  of  these  involution  forms  associated  with  typical 
bacilli  is  almost  diagnostic  for  one  with  experience.  Inoculating  tubes  of  plain  agar 
and  3%  salt  agar  with  this  same  material,  we  obtain  in  plain  agar  cultures  organisms 
which  are  typically  small,  fairly  slender  rods,  which  do  not  stain  characteristically 
at  each  end  and  are  not  oval.  The  smear  obtained  from  the  salt  agar  presents  most 
remarkable  involution  forms — coccoid;  root-shaped,  sausage-shaped  forms,  ranging 


PLAGUE    CULTURES 


97 


from  three  to  twelve  microns  in  length,  more  resembling  cultures  of  moulds  than 
bacteria.  Another  point  is  that  on  the  inoculated  plain  agar  we  are  in  doubt  at 
the  end  of  twenty-four  hours  whether  the  dewdrop-like  colonies  are  really  bacterial 
colonies  or  only  condensation  particles.  By  the  second  day,  however,  these  colonies 
have  an  opaque  grayish  appearance,  so  that  now,  instead  of  questioning  the  presence 
of  a  culture,  we  consider  the  possibility  of  contamination. 

Blood  cultures  in  septicsemic  plague  may  show  from  5  to  500,000  per  c.c.     Smears 
from  the  blood  in  such  cases  are  positive  in  only  about  17%. 


FIG.  31. — Pest  bacilli  from  spleen  of  a  rat.     (Kolle  and  Wassermann.} 

The  plague  bacillus  grows  well  at  room  temperature — -its  optimum 
temperature  being  30°  instead  of  37°  C.,  as  is  usual  with  pathogens. 
Next  to  the  salt  agar  culture,  the  most  characteristic  one  is  the  stalactite 
growth  in  bouillon  containing  oil  drops  on  its  surface.  The  culture 
grows  downward  from  the  under  surface  of  the  oil  drops  as  a  powdery 
thread.  These  are  very  fragile,  and  as  the  slightest  jar  breaks  them,  it 
is  difficult  to  obtain  this  cultural  characteristic. 

While  Klein  states  that  B.  coli,  proteus  vulgaris  and,  in  particular,  B.  bristolensis 
may  be  mistaken  for  plague  bacilli,  if  bipolar  staining  alone  be  relied  upon,  yet  it  is 
B.  pseudotuberculosis  rodentium  which  may  confuse  an  experienced  worker.  While 
this  latter  is  only  moderately  pathogenic  for  rats  yet  the  fact  that  rats  may  be 
immunized  to  B.  pestis  by  inoculation  with  B.  pseudotuberculosis  rodentium  brings 
up  the  suspicion  of  identity  of  the  two  organisms.  In  diagnosing  always  use  animal 
experimentation.  Owing  to  the  difficulty  in  emulsifying  plague  bacilli,  agglutination 
tests  are  not  satisfactory. 

Albrecht  and  Ghon  have  shown  that  by  smearing  material  upon  the 
intact,  shaven  skin  of  a  guinea-pig,  infection  occurs.  This  is  the  most 
crucial  test. 


g8  STUDY   AND    IDENTIFICATION   OF  BACTERIA 

A  pocket  made  by  cutting  the  skin  of  a  guinea-pig  with  scissors  and  extended 
subcutaneously  with  scissors  or  forceps,  into  which  a  piece  of  the  suspected  plague 
tissue  is  thrust  with  forceps,  is  more  practical  than  injecting  an  emulsion  with  hypo- 
dermic syringe. 

Mice  inoculated  at  the  root  of  the  tail  quickly  succumb.  Rats,  this  being  pri- 
marily a  disease  of  rats,  are  of  course  susceptible.  Other  rodents,  as  squirrels,  are 
susceptible.  It  has  been  suggested  that  a  rodent,  the  Siberian  marmot,  or  tarabagan 
(Arctomys  bobac)  might  be  the  starting-point  of  plague  outbreaks.  In  natural 
plague  of  rats,  the  lesions  which  establish  a  diagnosis  even  without  the  aid  of  a 
microscope  are  dark  red,  subcutaneous  injection  of  .the  flaps  of  the  abdominal  walls 
as  they  are  turned  back,  fluid  in  the  pleural  cavities,  cedematous  haemorrhagic 


FIG.  32. — Pest  bacillus  involution  forms  produced  by  growing  on  3%  salt  agar. 

(Kolle  and  Wassermann.) 

periglandular  infiltration  and  swelling  of  the  neck  glands,  and  in  particular  a  creamy, 
mottled  appearance  of  the  liver.  The  neck  glands  are  chiefly  involved  because  the 
flea  prefers  to  inhabit  the  skin  of  the  neck.  Smears  from  the  spleen  will  show  the 
oval  bacilli. 

A  chronic  rat  plague,  which  may  be  a  factor  in  keeping  up  the  disease,  is  char- 
acterized by  enlargement  of  the  spleen  and  the  presence  within  it  of  nodules  contain- 
ing plague  bacilli.  McCoy  has  noted  that  the  frequency  of  the  cervical  bubo  in 
rats,  noted  by  the  Indian  Commission  (72%),  was  not  found  in  California.  The 
glands  show  periglandular  infiltration  and  injection  as  well  as  enlargement. 

Recent  investigations  in  India  have  definitely  determined  the  fact 
that  the  flea  (Xenopsylla  cheopis)  is  the  intermediary  in  the  trans- 
mission of  plague  from  rat  to  rat  and  from  rat  to  man.  In  primary 
pneumonic  plague  the  infective  nature  is  very  great  and  appears  to  be 
by  the  respiratory  atrium  (From  man  to  man).  This  was  the  terri- 
fying type  of  plague  in  the  black  death  of  the  fourteenth  century. 


TYPHOID-COLON   GROUP  99 

Strong  and  Teague  have  shown  that  of  39  plates  exposed  before  the  mouths  of 
patients  with  pneumonic  plague,  with  marked  dyspnoea  and  pulmonary  oedema,  but 
without  coughing,  only  one  plate  showed  plague  bacilli.  In  39  other  experimental 
plate  cultures  with  coughing  on  the  part  of  the  patients  there  were  15  plates  showing 
plague  bacilli. 

The  droplet  method  of  infection  is  therefore  the  important  one  in  plague 
pneumonia. 

As  these  droplets  are  expelled  to  a  considerable  distance  not  only  should  the 
respiratory  inlets  be  protected  by  masks  but  the  conjunctivas  with  glasses  and 
abrasions  with  protective  coatings. 

For  diagnosis  make  smears  and  cultures  from  material  drawn  from 
a  bubo  by  a  syringe.  (At  a  later  stage,  when  softening  begins,  there  may 
not  be  any  bacilli  present.)  Also,  if  pneumonic  plague,  from  the 
sputum.  Blood  cultures  and  even  blood  smears  may  be  employed  in 
septicaemic  plague.  Formol  fuchsin  and  Archibald's  stain  make  satis- 
factory stains.  Always  inoculate  a  guinea-pig  with  the  material  either 
by  rubbing  it  in  with  a  glass  spatula  on  the  shaven  skin  or  by  sub- 
cutaneous injection.  For  prophylaxis  the  most  important  method  is 
that  of  Haffkine.  Stalactite  bouillon  cultures  of  plague  are  grown  for 
five  to  six  weeks.  These  are  killed  by  a  temperature  of  65°  C.  for  one 
hour.  Lysol  (1/4%)  is  added  to  the  preparation  and  from  0.5  to  4  c.c. 
injected,  according  to  the  age  and  size  of  the  individual  treated.  Sus- 
ceptibility is  reduced  about  one-fourth,  and  of  those  attacked  after 
previous  vaccination,  the  mortality  is  only  about  one-fourth  of  what  it  is 
among  the  noninoculated.  Strong  prepares  a  prophylactic  vaccine 
from  living  plague  cultures  rendered  avirulent.  Yersin's  serum,  made 
by  injecting  horses  with  dead  plague  cultures  and  afterward  with 
living  ones,  is  of  value  prophylactically  and  has  possibly  considerable 
curative  power. 

The  Eberth,  Gartner  and  Escherich  Groups. — From  a  standpoint 
of  cultures  in  litmus  milk  and  sugar  bouillon  we  can  divide  the  organ- 
isms related  to  typhoid  at  one  extreme  and  the  colon  at  the  other  into 
three  groups. 

i.  The  Eberth  or  typhoid  group.  There  are  three  important  patho- 
gens in  this  group:  the  B.  typhosus,  the  B.  dysenteriae,  and  the  3. 
faecalis  alkaligenes.  The  color  of  litmus  milk  is  practically  uhaltereU' 
and  there  is  no  gas  production  in  either  glucoSecr  lactose  bouillon.  Np 
coagulation  of  milk.  No  reduction' oi  ftcutral  red. '  The  J3.  typb'osiis. 
and  the  B.  faecalis  alkaligenes  are  actively  motile,  while'  the  B. dysen- 
teriae is  nonmotile  or  practically  so. 


100  STUDY   AND    IDENTIFICATION    OF   BACTERIA 

During  the  first  twenty-four  to  forty-eight  hours  there  is  a  moderate 
acid  production  by  typhoid,  so  that  the  milk  culture  is  less  blue,  while 
with  the  B.  faecalis  alkaligenes  the  alkalinity  is  intensified  from  the  start, 
so  that  the  blue  color  is  deepened. 

2.  The   Gartner   or  hog   cholera  group.     Besides  organisms  im- 
portant for  animals  and  probably  at  times  for  man,  such  as  B.  cholerae 
suum  and  B.  psittacosis  and  B.  icteroides  (interesting  historically  as 
having  been  reported  as  the  cause  of  yellow  fever  by  Sanarelli),  we  have 
two  pathogens:  i.  B.  enteritidis  (Gartner's  bacillus)  and  2.  B.  para- 
typhoid B.     In  this  connection  it  may  be  stated  that  the  present  view 
is  that  hog  cholera  is  caused  by  an  ultra-microscopic  organism  and  not 
by  the  B.  cholerae  suum. 

These  organisms  cannot  be  separated  culturally,  but  only  by  im- 
munity reactions.  They  do  not  turn  litmus  milk  pink.  They  produce 
gas  in  glucose  bouillon,  but  not  in  lactose.  They  very  powerfully  re- 
duce neutral  red  with  the  production  of  a  yellowish  fluorescence.  They 
do  not  coagulate  milk.  There  is  a  transient  acidity  in  the  litmus  milk, 
but  becoming  shortly  afterward  alkaline,  the  lilac-blue  color  is  intensi- 
fied. Both  organisms  are  motile. 

3.  The  Escherich  or  colon  group.     These  turn  litmus  milk  pink, 
coagulate  milk,  reduce  neutral  red,  and  show  varying  degrees  of  motility. 
The  three  groups  of  organisms  just  described  are  nonliquefiers  of  gela- 
tin.    Two  intestinal  organisms,  the  B.  cloacae  and  the  Proteus  vulgaris, 
differ  in  liquefying  gelatin. 

Bacillus  typhosus  (Eberth,  1880;  Gaffky,  1884).— This  organism 
may  be  isolated  from  the  stools,  urine,  and  the  blood  of  typhoid  patients. 

At  postmortem  it  can  be  best  isolated  from  the  spleen,  but  is  also  present  in 
Peyer's  patches  which  have  not  ulcerated.  When  ulceration  has  occurred  contami- 
nation with  B.  coli.  is  almost  sure.  Cultures  may  be  obtained  from  the  liver  also. 
In  sections  made  from  spleen  the  Gram  negative  bacilli  are  apt  to  be  decolorized. 
Thionin,  then  blotting  and  clearing  in  oil  or  xylol,  shows  the  clumps  of  bacilli  lying 
between  the  cells. 

Formerly  it  was  supposed  that  by  the  differences  in  the  thickness  of  the  film  of 
a  colony  or  by  its  varying  shades  of  grayish-blue,  we  possessed  data  of  importance  in 
differentiating  typhoid  from  related  organisms. 
?  ;Th£  cj&ipmes  look  like  grapevine  leaves. 

GrOwth *;en.,pota>x>w,as  A!S(?  considered  as  affording  information.  At  present, 
the,  biochemical  reactions  give  us-uucrroation  assisting  in  differentiation,  and  the 
agghVci nation '•  'aijd  bactpriolytic  phenomena,  the  final  diagnosis.  The  various 
plating  medfe/are  corisiderecl  Uftder- media  for  plating  out  faeces. 

Not  only  do  we  find  ny'pcrplasi'a  ef  rftfe/endothelial  cells  in  the  lymphoid  tissue 


TYPHOID    FEVER 


101 


of  Peyer's  patches  and  the  mesenteric  glands  and  the  spleen,  with  subsequent  necro- 
ses, but  focal  necroses  of  the  same  character  are  found  in  the  liver. 

A  striking  feature  of  the  pathology  of  typhoid  fever  is  the  long-con- 
tinued persistence  of  the  organisms  in  the  gall-bladder  and  elsewhere. 
It  is  beginning  to  be  believed  that  a  previous  typhoid  infection,  pos- 
sibly so  mild  as  to  have  passed  unnoticed,  is  at  the  basis  of  gall-bladder 
infections  and  resulting  gall-stones.  Various  bone  infections,  especially 
osteomyelitis,  have  shown  the  typhoid  bacilli  in  pure  culture.  For- 
merly it  was  supposed  that  the  typhoid  bacillus  brought  about  its  lesions 
by  a  local  infection  centered  in  the  ileum.  The  present  view  is  that 
typhoid  bacilli  effect  an  entrance  into  the  blood  stream  through  some 
lymphoid  channel,  as  by  tonsil  or  other  alimentary  lymphoid  structure. 
Of  animals,  only  the  chimpanzee  seems  to  be  susceptible. 


FIG.  33. — Seventy-two-hour-old  culture  of  typhoid  bacillus  on  gelatin.     (Kolle  and 

Wassermann.) 

They  develop  in  the  general  lymphatic  system,  the  spleen  in  partic- 
ular, where  they  are  protected  from  the  bactericidal  power  of  the  blood. 
After  a  time,  however,  approximately  the  period  of  incubation,  they 
become  so  abundant  in  these  lymphatic  organs  that  they  are  carried 
over  into  the  general  circulation.  Then  as  a  result  of  bacteriolysis  the 
intracellular  toxins  are  liberated  and  symptoms  develop.  If  bac- 
teriolysis takes  place  other  than  in  the  blood  we  have  various  suppurative 
processes.  As  a  result  of  the  formation  of  antibodies,  the  development 
in  spleen,  etc.,  is  checked  but  should  these  immunity  reactions  become 
less  potent  relapses  may  occur  or  various  local  infections  manifest 
themselves. 


102  STUDY   AND    IDENTIFICATION   OF  BACTERIA 

As  the  bacilli  do  not  multiply  to  any  extent  in  the  blood  itself  the  disease  cannot 
be  considered  as  a  typical  septicaemia  but  as  a  bacteriaemia. 

Typhoid  bacilli  can  be  isolated  from  the  blood  during  the  latter 
period  of  incubation  and  rarely  after  the  tenth  day  of  the  disease. 
It  is  a  practical  point  that  the  time  to  isolate  the  bacteria  from  the 
blood  is  in  the  first  days  of  the  attack.  The  diagnosis  by  agglutina- 
tion is  only  expected  after  the  seventh  to  tenth  day.  Agglutination 
may  not  appear  until  during  convalescence,  and  in  about  5%  of  the 
cases  it  is  absent.  It,  as  a  rule,  disappears  within  a  }tear. 

Very  little  success  has  b«en  obtained  with  curative  sera.  Chantamesse,  by 
treating  horses  with  a  nitrate  from  cultures  of  typhoid  bacilli  on  splenic  pulp  and 
human  defibrinated  blood,  claimed  to  have  obtained  a  curative  serum  possessing 
antitoxic  power.  Wright's  method  of  prophylactic  inoculation  is  now  being  em- 
ployed in  the  British  army  with  apparent  success.  In  this,  twenty-four-  to  forty- 
eight-hour-old  cultures  are  killed  at  53°  C.;  1/4%  of  lysol  is  then  added.  An  injec- 
tion of  500  million  bacteria  is  made  at  the  first  inoculation,  and  ten  days  later  an 
injection  of  one  billion.  The  British  prefer  to  inject  subcutaneously  in  the  infra- 
clavicular  region  and  at  the  insertion  of  the  deltoid.  The  Germans  consider  three 
injections  as  conferring  greater  immunity. 

Russell  has  obtained  splendid  results  in  the  U.  S.  Army  with  his  method  of 
vaccination.  In  this  three  injections  are  given  at  intervals  of  ten  days,  the  dosage 
being  500,000,000,  for  the  first  and  1,000,000,000  for  each  of  the  two  succeeding 
injections. 

Typhoid  vaccines  sterilized  with  0.5%  of  phenol  appear  to  keep  much  longer  and 
to  have  a  higher  immunizing  power  than  those  prepared  by  sterilization  with  heat 
and  subsequent  addition  of  the  antiseptic. 

Typhoid  bacilli  may  be  found  not  only  in  the  blood,  urine  and  faeces  but  as  well 
in  the  sputum  of  cases  showing  pulmonary  involvement.  They  have  also  been  found 
in  the  cerebrospinal  fluid  of  cases  showing  meningeal  symptoms.  At  the  autopsy 
they  may  be  found  in  the  spleen,  Pyer's  patches,  mesenteric  glands  and  liver. 

A  very  important  discovery  is  that  certain  persons,  who  may  have 
had  only  a  slight  febrile  attack,  may  eliminate  typhoid  bacilli  for  years 
in  their  faeces  (typhoid  carriers).  The  bacilli  are  also  eliminated  for 
considerable  periods  in  the  urine.  Distinction  is  now  being  made 
between  acute  carriers  (convalescents)  and  chronic  carriers. 

The  most  satisfactory  method  of  detecting  carriers  is  by  examina- 
tion of  faeces  or  urine  plated  out  on  Endo's  medium.  While  carriers 
usually  give  a  Widal  reaction  this  is  by  no  means  constant.  Typhoid 
carriers  are  said  to  maintain  a  high  opsonic  index. 

The  urine  and  faeces  of  typhoid  convalescents  should  be  proven  negative  by  cul- 
tural procedure  before  discharging J;hej3atients. 


TYPHOID    CARRIERS  103 

Vaccination  seems  to  be  a  very  satisfactory  measure  in  bringing  about  the  dis- 
appearance of  typhoid  bacilli  in  the  dejecta  of  carriers. 

For  laboratory  diagnosis,  blood  cultures  during  the  first  week  and 
agglutination  tests  during  the  second  week  and  onward  are  the  practical 
methods. 

Along  with  the,  agglutination  tests  the  urine  and  faeces  should  be  cultured  on 
Endo's  plating  medium  and  later  transferred  to  Russell's  medium  for  cultural 
identification.  The  positive  identification,  provided  the  culture  so  isolated  shows  the 
cultural  characteristics  of  typhoid,  is  made  by  testing  the  bacilli  for  agglutination 
with  a  known  typhoid  serum.  Instead  of  the  usual  blood  cultures  one  may  use  the 
clot  in  the  Wright  U-tube  for  culturing  and  the  serum  remaining  after  centrifugaliza- 


K$b 


FIG.  34.  —  Bacillus  of  typhoid  fever,  stained  by  Loffler's  method  to  show  flagella. 
(X  1000.)     (Williams.) 

tion  for  the  Widal  test  (clot  culture).  B.  typhosus  appears  in  the  blood  in  relapses. 
Kayser  considered  that  about  27%  of  cases  of  typhoid  in  Strasburg  were  caused  by 
raw  milk,  17%  by  contaminated  water,  17%  by  contact  with  typhoid,  and  10%  were 
due  to  typhoid  carriers.  Other  cases  were  due  to  infected  food,  and  about  13%  were 
of  origin  impossible  to  determine.  These  latter  may  have  been  due  to  unrecognized 
typhoid  carriers.  He  does  not  attach  the  same  importance  to  fly  dissemination  as 
do  American  authors. 

Contact  infection  is  the  great  factor  in  perpetuating  typhoid  fever 
but  this  agency  shows  diminishing  cases  each  year  provided  water  and 
milk  supplies  are  safe.  The  leading  European  cities  as  a  result  of  a  safe 


IO4  STUDY   AND    IDENTIFICATION    OF   BACTERIA 

water  supply  rarely  show  more  than  about  three  typhoid  deaths 
per  100,000  population  per  year.  Edinburgh  shows  less  than  one  per 
200,000  for  the  year  1910.  In  American  cities  rates  of  twelve  to  fif- 
teen per  100,000  are  common. 

The  Gartner  or  Meat-poisoning  Group. — Under  this  designation 
may  be  considered  the  organisms  which  cause  gastrointestinal  disorders 
of  varying  degrees,  infection  with  which  is  usually  brought  about  by  the 
ingestion  of  meat  obtained  from  diseased  cattle.  Unless  the  meat  is 
thoroughly  cooked  the  bacilli  in  the  interior  may  not  be  killed. 

In  this  group  may  be  placed  B.  enteritidis,  the  typical  meat-poisoning  organism, 
B.  paratyphoid  B,  B.  Danysz,  B.  Aertryck,  B.  typhi  muriiim  and  B.  suipestifer. 

B.  suipestifer  or  the  hog  cholera  bacillus  was  formally  thought  to  be  the  cause  of 
this  important  epizootic.  It  is  found  in  the  intestines  of  quite  a  percentage  of 
healthy  hogs.  The  cause  is  now  known  to  be  a  filterable  virus. 

These  organisms  are  alike  morphologically  and  culturally  and  show  quite  a 
tendency  to  bipolar  staining  and  reduction  of  neutral  red  with  fluorescence  in 
forty-eight  hours.  B.  paratyphoid  B,  B.  Aertryck  and  B.  suipestifer  are  alike 
from  an  agglutination  standpoint,  while  B.  enteritidis  and  B.  Danysz  show  similar- 
ity in  this  respect.  B.  paratyphoid  A  stands  by  itself. 

Paratyphoid  Bacilli  (Achard  and  Bensaude,  1896;  Schottmuller, 
1901). — Cases  resembling  mild  attacks  of  typhoid  occasionally  show 
agglutination  for  paratyphoid  bacilli.  These  organisms  have  also 
been  isolated  from  the  blood,  as  with  typhoid.  Two  types  have  been 
recognized:  the  paratyphoid  A  and  the  paratyphoid  B.  The  latter 
occurs  in  80%  of  such  cases.  Culturally,  paratyphoid  B.  cannot  be 
separated  from  Gartner's  bacillus.  In  paratyphoid  A  there  is  less  gas 
produced  in  glucose  bouillon  than  with  paratyphoid  B,  and  the  primary 
acidity  of  litmus  milk  is  not  succeeded  by  a  subsequent  alkalinity.  It 
does  not  seem  practical  to  draw  a  fine  distinction  between  these  two 
strains. 

Paratyphoid  B.  not  only  gives  symptoms  resembling  a  mild  typhoid  infection, 
but  may  show  symptoms  more  like  those  of  meat  poisoning  or  even  cholerine.  It 
is  more  pathogenic  for  laboratory  animals  than  is  B.  typhosus.  The  development  of 
antibodies  upon  immunizing  a  man  or  animal  with  paratyphoid  organism  does  not 
seem  to  approach  that  obtained  with  typhoid. 

Bacillus  enteritidis  (Gartner,  1888).' — This  organism  has  been 
frequently  isolated  from  cases  of  gastroenteritis  from  ingestion  of  in- 
fected meat. 

Meat  from  healthy  animals  which  has  been  in  contact  with  that  of  diseased 
animals  may  become  injected.  The  simple  act  of  placing  a  piece  of  infected  meat 


DYSENTERY  105 

on  a  sound  piece  may  infect  the  latter.  It  has  been  noted  that  the  bacteria,  or  their 
toxins,  may  be  distributed  unevenly  in  the  meat  eaten,  so  that  one  person  consuming 
the  same  meat  may  be  made  very  ill  while  others  eating  this  meat  may  escape 
infection.  Infection  of  food  may  occur  not  only  from  unclean  handling  but  from  the 
material  carried  by  flies  or  even  from  the  faeces  of  mice  or  rats  deposited  on  food- 
stuffs. 

This  organism  is  very  pathogenic  for  laboratory  animals,  producing  a  haemor- 
rhagic  enteritis  and  at  times  a  septicaemia.  Where  meat  has  been  contaminated  with 
Gartner's  bacillus  toxins  may  have  been  produced,  and  symptoms  of  poisoning  with 
acute  gastroenteritis  would  occur  shortly  after  ingestion.  This  is  not  a  true  toxin 
as  it  does  not  require  a  period  of  incubation  before  manifesting  its  toxic  action.  It 
is  interesting  to  note  that  this  toxin  is  not  destroyed  by  the  boiling  temperature,  thus 
differing  from  the  toxin  of  the  other  important  meat-poisoning  (botulism)  bacillus — 
B.  botulinus — which  is  rendered  innocuous  by  a  temperature  of  65°  or  70°  C.  If 
there  is  only  a  little  toxin  introduced  with  the  contaminated  meat,  the  symptoms 
will  be  delayed  one  or  two  days.  Such  organisms  have  been  isolated  in  pure  culture 
from  cases  with  high  fever,  marked  intestinal  derangement,  with  considerable  blood 
in  the  rather  fluid  stools.  In  two  cases  studied  the  disease  was  at  first  diagnosed 
as  a  severe  typhoid  infection.  Klein  thinks  the  organism  of  Danysz's  virus  (to 
kill  rats  during  plague  epidemics)  may  be  identical  with  B.  enteritidis. 

Proteus  vulgaris.— This  organism  is  often  encountered  in  plates 
made  from  faeces,  or  sewage  contaminated  water. 

It  is  common  in  decaying  meat  or  cheese,  and  cases  of  even  fatal  poisoning 
with  marked  gastrointestinal  symptoms  and  cardiac  failure  have  been  reported. 
At  times  it  is  the  cause  of  cystitis.  The  colonies  on  agar  are  moist  and  unevenly 
spreading  (amoeboid).  The  bacilli  are  very  motile,  long  and  slender,  tend  to  form 
filaments  and,  as  a  rule,  are  Gram  negative.  It  digests  blood-serum  and  is  a  rapid 
liquefier  of  gelatin.  In  litmus  milk  it  coagulates  with  a  soft  clot  and  an  alkaline 
reaction.  Subsequently  the  litmus  is  reduced  and  the  clot  digested  giving  a  dirty 
yellowish-brown  fluid.  Indol  is  rarely  produced.  The  cultures  generally  have  a 
putrefactive  odor.  In  infective  jaundice  (Weil's  disease)  this  organism  has  been 
reported  as  the  cause.  Organisms  of  this  group  were  formerly  designated  as  B. 
termo. 

Bacillus  dysenteriae  (Shiga,  1898). — -Dysentery  bacilli  produce  a 
coagulation  necrosis  of  the  mucous  membrane  of  the  large  intestine 
and  occasionally  of  the  lower  part  of  the  ileum.  Polymorphonuclears 
are  contained  in  the  fibrin  exudate. 

It  was  formerly  thought  that  these  lesions  were  of  local  origin,  but  the  present 
view  is  that  toxins  are  produced  which,  being  absorbed,  are  eliminated  by  the 
large  intestine  with  resulting  necrosis.  Flexner,  by  injecting  rabbits  intravenously 
with  a  toxic  autolysate,  produced  characteristic  intestinal  lesions.  The  toxin  with- 
stands a  temperature  of  70°  C.  without  being  destroyed.  The  toxin  may  cause 
joint  trouble. 


106  STUDY   AND    IDENTIFICATION   OF  BACTERIA 

There  are  two  main  types  of  dysentery  bacilli: 

1.  Those  producing  acid  in  mannite  media — 'the  acid  strains  (Flex- 
ner-Strong  types). 

2.  Those  not   developing  acid  in  mannite   (Shiga-Kruse   types). 
Ohno  finds  that  fermentative  reactions  do  not  correspond  to  immunity 
ones.     Thus  an  acid  strain  used  to  immunize  a  horse  may  produce  a 
serum  more  specific  for  a  nonacid  strain.     The  Shiga  type  is  very  toxic 
in  cultures,  while  the  Flexner  type  does  not  seem  to  possess  a  soluble 
toxin. 

The  Shiga  strains  are  apt  to  cause  a  paresis  of  the  hind  extremities  of  the  injected 
rabbit  which  may  be  followed  by  paralysis  and  death.  At  the  Lister  Institute 
injections  of  a  soluble  toxin  produced  a  serum  of  marked  antitoxic  power.  Such  a 
dysentery  serum,  which  is  probably  both  antitoxic  and  antimicrobic,  is  of  curative 
value.  Shiga  immunized  horses  with  polyvalent  cultures  and  obtained  a  polyvalent 
serum  which  has  reduced  the  death  rate  about  one-third. 

The  dysentery  bacillus  is  present  in  the  milky  white,  leukocyte  filled 
blood  flecked  mucous  stools  during  the  first  five  or  six  days  of  the  dis- 
ease. By  the  tenth  day  it  has  probably  disappeared.  Lactose  litmus 
agar  is  the  most  satisfactory  plating  medium.  The  stool  of  the  first 
two  days  may  give  practically  a  pure  culture.  The  staining  of  a  smear 
from  the  muco-purulent  stool  is  rich  in  phagocytic  cells,  many  of  them 
packed  with  Gram  negative  bacilli.  In  all  cultural  respects  the  dysen- 
tery bacillus  resembles  the  typhoid,  and  the  only  practical  method  of 
distinguishing  these  two  organisms,  other  than  by  agglutination  reac- 
tions, is  by  the  nonmotility  or  exceedingly  slight  motility  of  the  dysen- 
tery bacillus. 

The  characteristic  of  nonmotility  is  of  greatest  differentiating  value  and  the 
reports  of  slight  motility  are  probably  from  misinterpretation  of  molecular  movement 
as  motility.  The  dysentery  bacilli  do  not  form  those  threads  or  whip-like  filaments 
so  characteristic  of  typhoid  cultures  and  are  somewhat  plumper.  The  dysentery 
bacillus  is  not  found  in  the  blood  and  hence  is  not  eliminated  in  the  urine.  It  is 
found  in  mesenteric  glands.  In  dysentery  patients  agglutination  phenomena  do 
not  show  themselves  until  about  the  twelfth  day  from  the  onset.  Hence,  this 
procedure  is  of  no  particular  value  in  diagnosis.  It  is  of  value,  however,  to  identify 
an  organism  isolated  from  the  stools  at  the  commencement  of  the  attack,  using 
serum  from  an  immunized  animal  or  a  human  convalescent  for  the  agglutination 
test. 

Butler  has  suggested  taking  serum  from  dysentery  convalescents,  noting  the 
strain  involved,  and  preserving  it  by  taking  up  with  filter  paper  as  recommended 
by  Noguchi  for  the  Wassermann  haemolytic  amboceptor.  This  I  consider  very 


THE    COLON  BACILLUS  107 

valuable  as  it  is  very  difficult  to  immunize  rabbits  with  a  Shiga  strain  on  account  of 
its  great  toxicity. 

There  seems  to  be  very  little  agglutination  power  in  the  serum  of  convalescents 
from  Shiga  strains.  Flexner  strains  give  agglutination,  but  early  in  convalescence 
the  serum  is  not  apt  to  have  a  titre  of  more  than  1-50. 

Morgan  has  reported  as  the  cause  of  certain  cases  of  bacillary  dysen- 
tery a  bacillus  known  as  B.  Morgan,  No.  i.  It  is  motile,  produce  indol, 
and  in  glucose  bouillon  gives  a  very  slight  amount  of  gas. 

It  does  not  change  mannite  and  does  not  produce  a  primary  acidity  in  litmus 
milk.  This  organism  is  a  frequent  cause  of  summer  diarrhoea  of  children.  Flies 
from  houses  with  such  cases  often  show  Morgan's  bacillus.  A  dysentery  type  much 
like  the  Flexner  Strong  strain  is  often  found  in  the  enteric  affections  of  children  in 
the  United  States. 

In  Japan,  dysentery-like  epidemics  of  a  very  fatal  disease,  termed 
ekiri,  occur  among  young  children.  The  organism  is  very  motile,  pro- 
ducing gas  and  acid  in  glucose  but  not  in  lactose  media.  It  is  reported 
to  at  times  show  indol  production.  Apparently  a  member  of  the 
Gartner  group. 

More  recently  a  strain  of  dysentery  bacilli,  known  as  Type  F,  has 
been  considered  of  importance.  This  organism  is  very  closely  related 
to  the  Flexner  strain  and  only  differs  from  it  in  that  it  requires  about 
48  hours  to  turn  mannite  litmus  media  pink  and  that  maltose  litmus  re- 
mains blue.  An  organism  showing  similar  cultural  characteristics  has 
been  recently  recovered  from  faeces  of  laboratory  rabbits  by  German 
workers  investigating  the  problem  of  whether  certain  animals  might 
serve  as  carriers  for  dysentery. 

B.  COLI,  B.  LACTIS  AEROGENES,  B.  CLOACAE. 

While  the  COLON  BACILLUS  chiefly  inhabits  the  large  intestine,  the  B. 
lactis  aerogenes  is  to  be  found  in  the  upper  part  of  the  small  intestine. 
While  they  may  be  separated  on  the  ground  of  motility,  yet  it  is  by  the 
greater  fermentative  activity  of  the  B.  lactis  aerogenes  that  they  are 
best  separated.  Some  consider  them  as  only  representing  different 
strains  of  the  same  organism.  B.  lactis  aerogenes  is  closely  related  to  the 
pneumobacillus  and  at  times  shows  capsules.  Some  consider  that  the 
B.  coli  produces  a  bactericidal  substance  which  inhibits  the  growth  of, 
or  destroys,  pathogenic  bacteria  which  may  have  passed  the  destructive 
influences  of  the  gastric  juice;  others  that  this  effect  is  due  to  their  free 


108  STUDY   AND    IDENTIFICATION    OF   BACTERIA 

growth  and  the  development  of  phenol  and  various  putrefactive  sub- 
stances. The  probable  importance  of  the  colon  bacillus  in  protecting 
the  organism  is  shown  by  the  fact  that  where  numerous  colonies  of  patho- 
genic organisms  may  be  cultivated  from  fseces  we  may  find  a  diminu- 
tion in  number  or  absence  of  the  colon  bacillus.  This  condition  may  be- 
observed  in  infections  with  the  organisms  of  dysentery,  cholera,  typhoid, 
and  paratyphoid.  While  its  normal  function  is  probably  protective, 
yet  the  B.  coli  is  an  important  pathogenic  agent,  it  being  frequently  the 
organism  isolated  from  purulent  conditions  within  the  abdominal  cav- 
ity, especially  in  appendicitis  and  lesions  about  the  bile  ducts.  It  is 
particularly  prone  to  cause  lesions  of  the  bladder  and  pelvis  of  the  kid- 
ney. In  the  treatment  of  colon  cystitis  by  vaccines  of  dead  colon 
bacilli,  the  most  brilliant  results  in  opsonic  therapy  have  been  obtained. 

Sir  A.  Wright  thinks  that  certain  cases  of  mucous  colitis  may  be  due 
to  colon  infection  and  that  vaccination  may  cure  them.  The  colon 
bacillus  is  fully  considered  under  the  bacteriology  of  water. 

B.  CLOACA  was  isolated  first  from  sewage  by  Jordan.  It  is,  as  a  rule, 
a  rapid  liquefier  of  gelatin,  and  in  its  reactions  with  sugars  and  litmus 
milk  resembles  the  colon  bacillus. 

Where  the  gelatin  liquefaction  is  slow  or  slight  it  may  be  distinguished 
from  B.  coli  by  its  gas  formula  which  is  about  three  times  as  much  CO 2 
as  H,  just  the  reverse  of  that  of  the  colon  bacillus.  B.  lactis  aerogenes 
is  often  found  in  sewage.  It  is  one  of  the  causes  of  the  souring  of  milk. 

B.  ACIDOPHILUS,  B.  BlFIDUS,  B.  BULGARICUS. 

These  are  often  termed  the  long  rod  group  of  lactic  acid  bacteria 
in  contradistinction  to  certain  other  Gram  positive  bacilli  which 
are  short  and  oval  and  which  are  confused  with  the  so-called  milk 
streptococci. 

The  long  rod  group  often  forms  chains  and  often  shows  metachromatic  granules 
which  stain  with  Neisser's  method.  They  are'readily  distinguished  from  Gram  nega- 
tive lactic  acid  producers,  of  which  the  type  is  B.  lactis  aerogenes,  by  their  Gram 
positive  staining.  B.  acidophilus  often  give  the  impression  of  a  diphtheroid  in  a 
Gram  stained  faeces  smear.  It  is  nonmotile  and  often  shows  polar  granules.  Grows 
only  at  temperatures  above  22°  C.,  op.  37°  C.  It  grows  better  anaerobically  than 
aerobically  and  then  shows  the  clubbed  involution  characteristics  of  B.  bifidus; 
so  that  some  consider  these  organisms  the  same,  the  morphology  of  B.  bifidus  being 
the  result  of  anaerobiosis.  Original  cultures  are  best  made  in  i%  glucose  and  i% 
acetic  acid  bouillon.  Some  authorities  consider  B.  bifidus  the  most  important 


CHROMOGENIC   BACILLI  IOQ 

representative  of  the  large  intestine  flora.  B.  lactis  acidi  is  less  thermophilic  than 
B.  acidophilus  and  coagulates  milk  which  B.  acidophilus  does  not  do.  Certain 
polar  granule  bacteria,  as  B.  granulosum,  found  in  Yoghurt,  are  similar  to  B.  acido- 
philus but  coagulate  milk;  no  gas.  B.  bulgaricus  is  the  type  of  the  group  and  is 
discussed  under  milk. 

Rodella  thinks  B.  acidophilus,  B.  bifidus,  B.  gastrophilus  and  the  Boas-Oppler 
bacillus  identical.  B.  bulgaricus  is  said  to  never  show  polar  granules.  B.  bulgaricus 
and  the  group  of  organisms  similar  to  it  found  in  buttermilk,  etc.,  are  widely  used  in 
the  treatment  of  various  intestinal  troubles.  North  has  used  cultures  of  B.  bulgari- 
cus for  extermination  of  undesirable  organisms  in  other  parts  of  the  body  than  the 
alimentary  canal  (used  as  applications  in  nasal,  throat  or  geni to-urinary  infections). 


CHROMOGENIC  BACILLI. 

These  are  identified  by  the  color  of  their  colonies  on  agar.  The 
B.  pyocyaneus  is  the  most  important  one  of  them  in  medicine,  but  the 
B.  prodigiosus  is  also  of  interest  medically.  A  violet  chromogen,  the 
B.  violaceus,  which  is  motile  and  liquefies  gelatin,  has  been  described 
under  many  names.  It  has  been  found  in  water. 

An  orange-yellow  chromogen,  the  B.  fulvus,  is  nonmotile  and  varies 
as  to  its  liquefaction  of  gelatin. 

B.  pyocyaneus  (Gessard,  1882).— This  organism  is  frequently 
termed  the  bacillus  of  green  or  blue  pus.  It  is  a  small  (2.5X0.5/0 
motile  Gram  negative  bacillus. 

It  is  generally  a  slender  delicate  bacillus  often  showing  thread-like 
arrangement  but  at  times  it  may  appear  as  short  plump  rods.  It 
grows  readily  at  room  or  incubator  temperature.  It  liquefies  gelatin 
rapidly.  The  green  color  diffuses  through  the  agar  or  gelatin  on  which  it 
grows,  so  that  we  not  only  have  the  green-colored  colony,  but  the  me- 
dium as  well  is  colored.  Upon  potato  the  colonies  are  more  of  a  deep 
olive  green  to  dirty  brown. 

No  gas  is  produced  in  either  glucose  or  lactose  bouillon;  blood-serum  is  digested, 
the  pitted  surface  showing  a  reddish-brown  color.  The  protein  ferment  pyocyanase 
has  been  used  to  remove  diphtheritic  membrane  and  for  treatment  of  M.  catarrhalis 
nasal  catarrhs.  There  are  two  pigments — a  green  water  soluble  one  and  a  blue 
one  soluble  in  chloroform. 

It  is  widely  distributed  in  water  and  air,  and  is  frequently  isolated 
from  faeces.  The  B.  fluorescens  liquefaciens  of  water  seems  to  be  simply 
a  strain  of  B.  pyocyaneus.  The  B.  pyocyaneus  is  frequently  associated 
with  other  pus  organisms  in  abdominal  abscesses. 


110  STUDY   AND    IDENTIFICATION   OF  BACTERIA 

In  addition  to  having  an  endotoxin,  it  produces  a  soluble  toxin  similar  to  diph- 
theria toxin.  This  toxin  differs  from  those  of  diphtheria  and  tetanus  in  that  it  can 
stand  a  temperature  of  100°  C.,  while  those  of  diphtheria  and  tetanus  are  destroyed 
at  about  65°  C.  The  fact  that  the  union  between  toxin  and  antitoxin  is  only  of  a 
binding,  neutralizing  nature  is  best  shown  by  taking  a  mixture  of  pyocyaneus  toxin 
and  antitoxin  which  is  innocuous  and  heating  it.  This  destroys  the  antitoxin,  but 
does  not  injure  the  toxin.  We  now  find  that  the  original  toxicity  has  returned.  The 
antitoxins  of  diphtheria  and  tetanus  are  more  stable  than  the  corresponding  toxins; 
hence,  this  experiment  would  be  impossible  with  them,  as  upon  heating  we  should 
first  destroy  the  toxin. 

On  account  of  the  frequent  association  of  B.  pyocyaneus  with  other 
organisms  of  better  recognized  pathogenicity  it  has  until  more  recently 
been  considered  rather  harmless;  this  view  can  no  longer  be  entertained 
as  it  is  frequently  the  sole  cause  of  middle-ear  inflammations,  intestinal 
disorders  and  possibly  at  times  of  septicaemia. 


FIG.  35. — Bacillus  pyocyaneus.     (Kolle  and  Wassermenn.) 

B.  prodigiosus. — 'This  is  a  very  small  coccobacillus  which  shows  mo- 
tility  in  young  bouillon  cultures.  It  is  Gram  negative.  The  colonies 
on  agar  or  other  solid  media  show  a  rich  red  color.  The  pigment  only  de- 
velops at  room  temperature;  it  is  absent  in  cultures  taken  out  of  the 
incubator.  The  B.  prodigiosus  is  frequently  found  on  foodstuffs, 
especially  bread,  where  it  may  simulate  blood.  It  liquefies  gelatin  rap- 
idly and  gives  a  diffuse  turbidity  to  bouillon.  It  is  probable  that  B. 
indicus  and  B.  kilensis  are  strains  of  B.  prodigiosus. 

Coley's  fluid,  which  has  been  used  in  cases  of  inoperable  sarcoma  and  other 
malignant  growths,  is  a  culture  prepared  by  growing  very  virulent  streptococci  in 


COLEY'S  FLUID  in 

bouillon  for  ten  days.  This  streptococcus  culture  is  now  inoculated  with  B.  prodi- 
giosus,  and  after  another  ten  days  the  mixed  culture  is  killed  by  heat  at  60°  C.  and  the 
sterile  product  injected.  Coley  injected  about  one-twentieth  of  a  c.c.  of  this  vaccine. 
At  present  he  uses  nonnltered,  heat  sterilized  bouillon  cultures  of  a  streptococcus 
obtained  either  from  a  case  of  erysipelas  or  septicaemia.  To  this  is  added  material 
from  agar  cultures  of  B.  prodigiosus,  grown  separately  and  sterilized  before  adding 
to  the  sterilized  streptococcus  bouillon  culture. 


CHAPTER  IX. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.     SPIRILLA. 
KEY  AND  NOTES. 

KEY  to  recognition  of  gelatin  liquefying,  motile  and  Gram  negative 
spiral  or  comma-shaped  organisms. 

A.  Do  not  give  the  nitroso-indol  reaction  with  sulphuric  acid  alone  in  twenty-four 
hours. 

1.  Produce  an  abundant  moist  cream-colored  growth  on  potato  at  room  tempera- 
ture. 

a.  Finkler  and  Prior's  spirillum  (Vibrio  proteus).  Liquefaction  of  gelatin 
very  rapid.  No  air-bubble  appearance  at  top  of  liquefied  area.  Cultures 
have  foul  odor.  Milk  coagulated.  Thicker  spirillum  than  cholera.  Iso- 
lated from  cholera  nostras. 

2.  Scanty  growth  or  none  at  all  on  potato  at  room  temperature.     Only  a  mod- 

erate yellowish  growth  when  incubated  about  incubator  temperature, 
a.  Spirillum  tyrogenum  (Deneke's  spirillum).     Does  not  liquefy  gelatin  so 
rapidly  as  Finkler  Prior.     Thinner  and  smaller  spirillum  than  cholera. 

B.  Give  the  nitroso-indol  reaction  with  sulphuric  acid  within  twenty-four  hours. 

1.  Very  pathogenic  for  pigeons. 

a.  Spirillum  metschnikovi.  Liquefies  gelatin  about  twice  as  rapidly  as 
cholera.  Gives  bubble  appearance  at  top  of  stab. 

2.  Scarcely  pathogenic  for  pigeons, 
a.  Spirillum  choleras  asiaticae. 

Nonmotile,  nonliquefying  and  Gram  positive  spirilla  have  also  been  described. 
There  is  also  a  large  group  of  phosphorescent  spirilla. 

Spirillum  cholerae  asiaticse  (Koch,  1884).— Typically,  the  mor- 
phology of  this  organism  is  that  of  the  comma  (Comma  bacillus  of 
Koch).  It  also  frequently  shows  S  shapes,  and  often  appears  in  long 
threads  showing  turns.  When  freshly  isolated  from  cholera  material 
they,  as  a  rule,  show  a  fairly  typical  morphology,  but  after  subcultures 
in  the  laboratory  variations  are  common,  so  that  rod  forms  and  round 
involution  shapes  give  a  picture  altogether  at  variance  with  the  comma 
shape. 

Even  in  recent  cultures  of  undoubted  cholera  we  may  have  different  types,  as 
coccoid  forms  and  slender  rods.  Ohno  has  noted  the  fact  that  the  same  strain  of 
cholera  will  give  at  one  time  vibrio  forms  and  again  coccoid  or  rod  forms,  depending 

112 


CHOLERA 


on  the  reaction  of  the  media.  Inasmuch  as  the  recognition  of  vibrio  shapes  is  of 
importance  in  diagnosis  he  recommends  that  material  from  a  stool  be  inoculated 
into  three  tubes  of  peptone  solution  of  reaction  +0.3,  —0.5  and  —  i,  respectively,  one 
of  which  would  probably  show  vibrio  morphology. 


FIG.  36. — Cholera  spirilla.     (Kolle  and  Wassermann.) 

The  cholera  spirillum  is  very  motile  (a  scintillating  motility)  and 
liquefies  gelatin  fairly  rapidly,  although  more  slowly  than  any  of  the 
spirilla  mentioned  in  the  key.  The  colony  on  gelatin  was  formerly 
considered  characteristic,  but  like  most  cultural  characteristics,  it  is  now 


FIG.  37. — Involution  forms  of  the  spirillum  of  cholera.     (Van  Ermengen.} 

considered  as  being  only  of  confirmatory  value;  it  is  not  specific.  These 
colonies  show  in  twenty-four  hours  as  small  granular  white  spots  which 
have  a  spinose  periphery.  An  encircling  ring  of  liquefaction  now 
makes  its  appearance  and  the  highly  refractile  (as  if  fragments  of  spark- 

8 


STUDY   AND    IDENTIFICATION   OF  BACTERIA 


ling  glass)  colony  can  be  separated  into  a  granular  center,  a  striated 
periphery,  and  a  clear  external  ring  of  liquefaction. 

On  gelatin  stabs  the  liquefaction  produces  a  turnip-like  hollow  at  the  top  of  the 
puncture — the  air  bubble  appearance.  It  gives  the  nitroso-indol  reaction  with  sul- 
phuric acid  alone  (cholera  red).  Kraus  attaches  importance  to  the  fact  that  cholera 
does  not  produce  a  haemolytic  ring  on  blood  agar  as  do  the  pseudocholera  spirilla; 
a  difficulty  is  that  many  pseudospirilla  do  not  haemolize.  Furthermore,  true  cholera 
strains  may  occasionally  show  haemolysis,  especially  in  laboratory  cultures.  Quite 
a  discussion  has  arisen  in  connection  with  a  spirillum  isolated  from  cases  of  diarrhoea 
(no  symptoms  of  cholera)  in  pilgrims  at  El  Tor.  This  organism  gave  the  immunity 
reactions  (agglutination)  of  true  cholera  but  on  account  of  its  haemolytic  power  has 
been  considered  as  distinct  from  cholera.  Such  a  view  would  seem  to  be  untenable. 
Sp.  choleras  grows  very  rapidly  on  peptone  solution  and  this  is  the  medium  for  the 
enrichment  test  to  be  later  described.  On  this  it  may  form  a  pellicle.  On  agar 
the  colony  is  more  opalescent  (more  of  a  translucent  grayish  blue)  than  the  typhoid. 
It  does  not  grow  on  potato  except  at  incubator  temperature.  It 
does  not  coagulate  or  turn  acid  litmus  milk.  The  spirilla  are 
found  in  myriads  in  the  rice-water  discharges,  these  white 
flakes  being  desquamated  epithelial  cells.  They  penetrate  the 
crypts  of  Lieberkuhn,  but  rarely  extend  to  the  submucosa. 
The  symptoms  are  due  to  an  endotoxin. 

Cholera  may  be  transmitted  from  water  supplies, 
when  the  outbreak  is  apt  to  be  widespread  and  in 
great  numbers  from  the  start.  Also  by  indirect  con- 
tagion, as  by  flies  or  on  lettuce,  etc.  A  very  im- 
portant point  is  that  we  have  well  persons  whose  faeces 
contain  virulent  cholera  spirilla  (cholera  carriers). 

Cholera  spirilla  disappear  from  the  stools  of  cholera 
patients  very  rapidly,  usually  in  five  to  ten  days. 
Cholera  carriers  are  therefore  of  less  importance  epi- 
riUum  of  cholera  demiologically  than  typhoid  carriers, 
stab  c  u  1 1  u  r  e  in  jt  js  we^  to  remember  however  that  cases  have  been  reported 
oW.atl CFrTe n kll  of  Positive  findings  after  a  period  approximating  two  months 
and  Pjeifer.}  from  the  onset  of  the  attack  of  cholera.  Another  important 

consideration  is  that  the  vibrios  may  be  absent  at  one  examina- 
tion and  be  present  at  a  later  one.  Purgatives  seem  to  influence  the  reappearance 
of  the  spirilla.  An  acid  reaction  of  the  faeces,  such  as  that  induced  by  lactic  acid 
bacteria,  would  apparently  be  of  value  in  the  prophylaxis  of  cholera  carriers. 

Greig  has  found  infection  of  the  bile  of  the  gall-bladder  or  ducts  in  80  cases  in 
271  cholera  autopsies.  While  cholera  spirilla  are  soon  crowded  out  by  intestinal 
bacteria,  thus  explaining  the  short  period  during  which  cholera  spirilla  are  excreted 
by  convalescents,  this  is  not  true  when  the  cholera  vibrio  gets  into  the  bile  ducts  or 
gall-bladder,  where  ideal  conditions  prevail  for  a  prolonged  life.  In  fact  bile  has 


FIG.    38. — Spi- 


CHOLERA   DIAGNOSIS  1 15 

recently  been  recommended  as  a  selective  medium  for  cholera  enrichment.  Greig 
found  one  cholera  convalescent  excreting  cholera  vibrios  44  days  after  the  attack. 
Of  twenty-seven  persons  who  had  been  in  contact  with  cholera  patients  six 
were  excreting  cholera  vibrios  though  apparently  well. 

To  identify  such  spirilla  immunity  reactions  are  necessary: 

1.  Injected  intraperitoneally  into  guinea-pigs,  it  produces  a  perit- 
onitis   and    subnormal   temperature.     This  reaction   exists    for 
spirilla  other  than  the  true  cholera  spirillum. 

2.  Intramuscular  injections  into  pigeons  are   only  slightly  patho- 
genic, if  at  all. 

3.  The  agglutination  test  is  the  most  practical.     In  this  we  use  serum 
from  an  immunized  animal,  in  dilution  of  from  100  to  1000.     It  is 
rare  that  true  cholera  vibrios  fail  to  agglutinate  in  serum  of  i  to 
500  and  even  sera  of  i  to  10,000  dilution  give  the  reaction.     Serum 
of  cholera  convalescents  may  show  agglutination  as  early  as  the 
tenth  day;  it  is  usually  best  shown  about  the  third  week.     Dunbar's 
quick  method  is  very  practical.     Make  two  hanging-drop  prepa- 
rations, using  mucus  from  the  stool  as  the  bacillary  emulsion.     To 
one  add  an  equal  amount  of  a  i  :  50  normal  serum;  to  the  other  a 
i  :  500  dilution  of  immune  serum.     Cholera  spirilla  remain  motile 
in  the  control,  but  lose  motility  and  become  agglutinated  in  the 
preparation  with  the  immune  serum. 

4.  Pfeiffer's  phenomenon.     If  cholera  spirilla  are  introduced  into  the 
peritoneal  cavity  of  immunized  guinea-pigs  (or  if  together  with  a 
i  :  1000  dilution  of  immune  serum  the  mixture  is  injected  intra- 
peritoneally into  normal  guinea-pigs)  and  at  periods  of  ten  to 
sixty  minutes  after  injection,  material  is  removed  by  a  pipette  from 
the  peritoneal  cavity,  the  spirilla  have  lost  motility,  have  become 
granular  and  degenerated.     Pseudospirilla  are  unchanged.     This 
reaction  may  be  carried  on  in  a  pipette,  using  fresh  serum. 

Antisera  for  the  treatment  of  cholera  have  not  proved  successful.  Prophylac- 
tically,  there  are  two  prominent  methods:  i.  That  of  Haffkine,  where  live  cholera 
spirilla  are  injected  subcutaneously;  and  2.  Strong's  cholera  autolysate.  In  this 
cholera  cultures  are  killed  at  60°  C.  The  killed  culture  is  then  allowed  to  digest 
itself  in  the  incubator  at  37°  C.  for  three  or  four  days  (peptonization).  The  prepara- 
tion is  then  filtered  and  from  2  to  5  c.c.  of  the  filtrate  is  injected.  Ferran  was  the 
first  to  use  vaccines. 

For  diagnosis :  i .  take  a  fleck  of  mucus,  make  a  straight  smear  and 
fix;  stain  with  a  i  :  10  carbol  fuchsin.  The  comma-shaped  organisms 
appear  as  fish  swimming  in  a  stream. 


Il6  STUDY    AND    IDENTIFICATION    OF   BACTERIA 

2.  Inoculate  a  tube  of  peptone  solution.     The  cholera  spirilla  grow 
so  rapidly,  and  being  strong  aerobes,  they  grow  on  the  surface  of 
the  fluid  so  that  by  taking  a  loopful  from  the  surface,  we  may  in 
three  to  eight  hours  obtain  a  pure  culture.     Should  there  be  a 
pellicle  present,  this  should  be  avoided  in  the  transfer  by  tilting 
the  tube  slightly,  so  that  the  material  near  the  surface  be  obtained 
without  touching  the  pellicle.     Inoculate  a  second  tube  from  the 
surface  of  this  first  and,  if  necessary,  a  third  (enrichment  method). 

3.  Test  for  cholera  red  reaction.     (Simply  adding  from  three  to  five 
drops  of  concentrated  chemically  pure  sulphuric  acid  to  the  first 
or  second  peptone  culture  after  eighteen  to  twenty-four  hours' 
growth.     Some  specimens  of  peptone  do  not  give  the  reaction.) 
At  times  we  only  get  the  cholera  red  when  we  have  a  pure  culture 
of  cholera. 

4.  Smear  a  fleck  of  mucus  or,  better,  the  three  hour  surface  growth 
of  a  peptone  culture  on  a  dry  agar  surface  in  a  Petri  dish.     From 
colonies  developing,  make  agglutination  and,  if  desired,  cultural 
tests.     It  is  by  immunity  reactions  that  we  identify  cholera  spirilla. 
The  surface  moisture  of  plates  is  best  dried  by  the  filter-paper  top. 

The  cholera  colony  is  easily  distinguished  from  the  ordinary  faecal  bacterial 
colonies  by  its  transparent,  bluish-gray,  delicate  character.  It  emulsifies  with  the 
greatest  ease.  A  practical,  quick  method  is  to  make  smears  from  suspicious  colonies, 
stain  for  one  minute  with  dilute  carbol  fuchsin  and  if  vibrios  are  present  to  make 
two  vaseline  rings  on  a  single  slide  allowing  ample  space  at  one  end  for  handling  the 
preparation  safely.  Inside  of  one  ring  deposit  with  a  platinum  loop  a  drop  of  salt 
solution  and  inside  the  ring  nearest  the  end  which  is  to  be  held  by  fingers  or  forceps, 
deposit  a  loopful  of  i  to  500  or  i  to  1000  dilution  of  cholera  serum.  The  emulsion 
in  the  salt  solution  remains  uniformly  turbid  and  under  a  low  power  of  the  microscope 
(2/3  in.)  shows  a  scintillating  motility.  The  emulsion  made  into  the  drop  of  serum 
quickly  shows  a  curdy  agglutination  and  upon  examination  with  the  2/3-in.  objective 
shows  clumping  and  absence  of  motility.  Cover-glasses  placed  over  the  two 
vaseline  rings  assist  in  the  study  of  the  preparation. 


CHAPTER  X. 


STUDY  AND  IDENTIFICATION  OF  MOULDS. 

CLASSIFICATION  or  THE  FUNGI. 


Order  Suborder 

Phycomycetes      Zygomycetes 


Ascomycetcs 


Gymnoascus 


.  Carpoascus 


Family               Genus 

Species 

JVtticor                •  -, 

M.  corymbifer 

^  M.  mucedo 

Rhizomucor 

R.  septatus 

Rhizopus 

R.  niger 

!S.  cerevisiac 

Saccharomyces     ' 

S.  anginas 

S.  blanchardi 

Saccharo- 

mycetes 

Endomyces 

E.  albicans 

Cryptococcus 

r  C.  gilchristi 
C.  hominis 

T.  sabouraudi 

T.  tonsurans 

r  Trichophyton       < 

T.  violaceum 

T.  mentagro- 

Gymno- 

phytes 

asceae 

T.  cruris 

, 

Microsporum 

M.  audouini 

,  Achorion 

A.  schoeleini 

Penicillium 

P.  crustaceum 

A.  fumigatus 

Perisporia-    , 

Aspergillus 

A.  concentricus 

ceae 

A.  pictor 

A.  niger 

Discomyces 

D.  bo  vis 
k  D.  madurae 

Madurella 

M.  mycetomi 

JMalassezia 

M.  furfur 

Microsporoides 

M.  minutissi- 

mus 

Trichosporum 

T.  giganteum 

Sporotrichum 

S.  beurmanni 

Hyphomycetes 


NOTE. — In  many  of  the  works  on  bacteriology  considerable  space  is  given  to 
the  so-called  Higher  Bacteria.  The  organisms  are  chiefly  considered  under  the  names 
Leptothrix  or  forms  in  which  are  found  simple  nonbranching  threads,  Cladothrix 
or  thread-like  forms  with  false  branching  and  Streptothrix  or  forms  showing  true 
branching.  It  is  not  practical  to  consider  any  separate  group  distinct  from  the  so- 
called  Lower  Bacteria  on  the  one  hand  and  the  Fungi  on  the  other. 

117 


Il8  STUDY   AND    IDENTIFICATION    OF   MOULDS 

•  i 

THE  FUNGI. 

The  Thallophyta  are  plants  in  which  there  is  no  differentiation  be- 
tween root  and  stem. 

The  classes  of  Thallophyta  which  are  of  interest  medically  are  i .  the 
Algae  and  2.  the  Fungi. 

Some  include  Lichenes  as  a  separate  class.  These  are  really  sym- 
biotic organisms — 'Fungi  parasitic  on  Algae. 

The  Algae  contain  chlorophyll,  with  the  exception  of  Cyanophyceae. 
To  the  order  Cyanophyceae  it  is  considered  that  the  family  of  bacteria 
belong. 

The  fungi  do  not  possess  chlorophyll.  They  are  in  their  simplest  forms  ramifying 
filaments  called  hyphse.  The  vegetative  hyphae  which  intertwine  in  tangled  threads, 
as  a  support,  are  termed  the  mycelium,  while  those  which  project  upward  are  called 
the  aerial  hyphae  and  are  the  ones  which  bear  the  conidia  or  spores. 

The  aerial  hypha  which  carries  the  fruiting  organ  encasing  the  conidia  (sporan- 
gium) is  called  the  sporangiophore  and  the  more  or  lesg  rounded  termination  of  this 
hypha,  which  projects  into  the  sporangium,  is  called  the  columella. 

The  hypha  may  be  composed  of  one  cell  or  of  many  cells  separated  by  septa 
(septate). 

The  orders  of  the  class  Fungi  which  are  of  interest  medically  are: 
i.  the  Phy corny cetes;  2.  the  A<comycetes;  3.  the  Hyphomycetes. 

Phycomycetes. — These  produce  a  copious  network-like  mycelium,  which  is  non- 
septate,  and  reproduce  asexually  by  means  of  a  sporangium,  a  case-like  structure 
borne  on  the  clubbed  extremity  of  an  erect  hypha  (columella)  and  containing  numer- 
ous spores  or,  as  in  the  case  of  the  suborder  Oomycetes,  reproduction  is  by  hetero- 
gamy.  (Dissimilar  sexual  cells — a  smaller  male,  antheridium,  and  a  larger  female, 
oogonium.  By  fertilization  by  antherozoids  from  the  antheridium  penetrating  the 
oosphere  we  have  oospores.) 

The  suborder  Zygomycetes  reproduces  either  asexually  (a  sporangium  filled  with 
spores)  or  by  isogamy  (two  similar  but  sexually  differentiated  cells  conjugate  and 
form  on  fusion  a  zygospore). 

Belonging  to  this  suborder  we  have  four  families,  only  one  of  which,  the  Mucor 
family,  is  of  importance  medically.  In  this  family  we  have  three  genera:  Mucor, 
without  rhizoids;  Rhizopus,  with  rhizoids  and  unbranched  aerial  hyphae  and, 
Rhizomucor,  with  rhizoids  and  ramified  mycelium. 

Two  species  of  Mucor  are  of  pathogenic  importance. 

i.  Mucor  mucedo  and  2.  Mucor  corymbifer.  These  moulds  develop  especially 
in  external  cavities  as  nasopharynx  and  external  ear. 

Pulmonary  and  generalized  infections  have  also  been  reported.  The  pathogenic 
species  have  smaller  spores  and  grow  best  at  37°  C.  The  thick,  coarse,  cotton-like 
mould  seen  on  horse  manure  is  a  Mucor.  The  sporangium,  the  organ  of  fructifica- 


YEASTS  IIQ 

tion,  contains  the  spores  within  its  interior.  The  M.  mucedo  has  thick  silver-gray 
mycelium,  with  large  sporangia,  150 ft  in  diameter,  containing  oval  spores,  5  X  Qj". 
The  M.  corymbifer,  which  has  been  reported  from  a  generalized  infection,  con- 
sidered as  typhoid,  shows  a  snow-white  mycelium.  The  sporangia  are  20  to  4o/x 
and  the  spore  about  3jw  in  diameter. 

Rhizopus  niger  has  a  columella  which  becomes  distorted  into  a  mushroom  shape 
after  the  spores  have  been  discharged  from  the  sporangium.  This  mould  has  been 
considered  as  the  cause  of  a  mycosis  of  the  tongue. 

Ascomycetes. — -In  this  order  are  included  many  of  the  parasitic 
moulds.  The  most  distinctive  characteristic  is  the  formation  of  asco- 
spores  in  an  ascus  (little  sac).  It  is  an  enlarged  extremity  of  a  hyphal 
branch  in  which  a  definite  number  of  spores,  usually  eight,  is  formed. 
The  ascus  usually  ruptures  at  its  tip.  Other  members  of  the  order  are 


FIG.  39. — Yeast  cells.     Saccharomyces  cerevisiae.     (Coplin.) 

formed  from  hyphae  by  the  separation  of  cells  in  succession  from  the 
free  cells.     The  mycelium  is  septate. 

The  order  is  divided  into  those  with  naked  asci  (Gymnoascus)  and  those  having 
a  perithecium  or  investing  layer  about  the  ascus  (Carpoascus). 

Belonging  to  the  suborder  Gymnoascus  we  have  i.  the  family  of  Saccharomycetes, 
which  reproduce  by  budding  and  in  which  the  asci  are  without  any  semblance  of  a 
sheath,  and  2.  a  family  in  which  there  is  an  indication  of  the  formation  of  a  peri- 
thecium— this  may  be  termed  the  Gymnoasceae  family. 

Saccharomycetes. — There  are  three  genera:  Saccharomyces,  Endomyces,  and 
Cryptococcus. 

Saccharomyces. — These  reproduce  by  budding,  have  ascopores  and  no  mycelial- 
like  threads. 

S.  cerevisiae. — This  is  the  ordinary  yeast  fungus.     Used  at  times  as  an  antiseptic. 
S.  anginae. — Found  in  a  case  of  angina. 


120  STUDY   AND    IDENTIFICATION    OF    MOULDS 

S.  blanchardi. — Found  in  a  jelly-like  tumor  mass  of  the  abdomen.  The  budding 
cells  varied  from  2  to  20/1.  Probably  identical  with  S.  tumefaciens,  reported 
as  the  cause  of  a  subcutaneous  tumor  about  region  of  Scarpa's  triangle. 
Endomyces. — Forms  spores  in  the  interior  of  filaments,  or  by  ascus  formation  or  by 
chlamydospores  (resistant  spore-like  structures  with  a  thick  membrane  which 
project  from  the  extremities  or  sides  of  the  hyphae  as  bud-like  structures). 

E.  albicans. — The  organism  of  thrush.  It  produces  a  false  membrane,  especially 
on  buccal  surfaces,  which  is  easily  detached  and  beneath  which  the  mucosa  is 
intact.  Grows  only  in  acid  media.  Hence  propriety  of  alkaline  treatment. 
Cryptococcus. — Reproduces  by  budding,  but  ascospore  formation  not  observed. 
Not  a  well-recognized  genus.  The  diseases  caused  by  it  are  termed  blasto- 
mycoses. 

C.  Gilchristi. — The  cells  are  about  i6/*  in  diameter  and  have  a  thick,  double 
contoured  membrane.  They  reproduce  by  budding.  The  skin  lesions  resemble 
various  infectious  granulomata  and  diagnosis  rests  on  the  finding  of  budding 


FIG.  40. — Thrush  fungus.     (Kolle  and  Wassermann.) 

or  sporulating  cells.  It  may  invade  internal  organs.  Original  cultures  are 
obtained  with  some  difficulty  and  then  best  with  LofHer's  serum.  Subcultures 
grow  readily.  Potato  is  a  good  medium  and  on  it  we  may  have  both  mycelial 
and  yeast-like  growth.  Guinea-pigs  can  be  inoculated  subcutaneously.  A 
mould,  somewhat  similar,  is  the  Coccidioides  immitis  of  Ophuls.  This  has  a 
mycelial  growth  in  tissues,  this  distinguishing  it  from  the  former  fungus.  The 
infection  frequently  becomes  generalized.  The  small  bodies,  about  3jw,  in  the 
Molluscum  contagiosum  cells  are  thought  by  some  to  be  yeasts.  They  are  more 
probably  artefacts.  Plimmer's  bodies  in  cancer  cells  belong  in  this  group. 
They  also  are  probably  other  than  parasites. 

C.  linguae  pilosae. — This  is  a  more  or  less  elongated  yeast-like  organism  and  sup- 
posed to  be  the  cause  of  black  tongue,  a  benign  affection  of  the  lingual  papillae. 

Gymnoascea. — -Belonging   to  the  family  Gymnoasceae  we  have  the 
genera  Trichophyton,  Microsporum  and  Achorion. 


RINGWORM 


121 


The  trichophytons  are  generally  known  as  the  large-spored  ring-worms.  The 
spores  are  in  chains  and  may  be  inside  the  hair  or  both  outside  and  inside.  Many 
of  them  are  of  animal  origin,  especially  from  the  horse  and  the  cat.  The  spores  are 
from  5  to  7/1. 

The  mycelium  is  greatly  segmented,  shows  simple  or  dichotomous  branching, 
and  produces  spores  within  the  mycelium. 

T.  tonsurans. — Gives  a    crater-like  culture  with  fine  marginal  rays.     Fungus 
wholly  inside  the  hair.     Causes  most  of  the  large-spored  scalp  ringworms  and 
many  body  cases. 
T.  sabouraudi. — Has  a  heaped-up  festooned  sort  of  culture.     There  is  a  similar 


10. 


FIG.  41.- — More  common  fungi,  i,  Culture  of  Achorion  schoenleini  (favus); 
2,  culture  of  Trichophyton  tonsurans;  3,  culture  of  Trichophyton  sabouraudi;  4, 
sporangium  of  Aspergillus;  5,  culture  of  Trichophyton  mentagrophytes;  6,  culture 
of  Microsporum  audouini;  7,  mycelium  and  spores  of  Malassezia  furfur;  8,  Crypto- 
coccus  gilchristi;  9,  A  and  B,  sporangium  and  mycelium  of  Mucor  corymbifer;  10, 
Penicillium;  n,  Saccharomyces  tumefaciens;  12,  Discomyces  bovis. 

fungus  with  a  violet  culture.     These  cause  some  of  the  scalp  and  beard  ring- 
worms. 

T.  mentagrophytes. — This  is  the  T.  megalsporon  endoectothrix  of  Sabouraud. 
The  external  spores  are  in  chains  or  in  short  mycelial  threads,  not  mosaics  of 
spores,  and  are  of  very  unequal  size  (2  to  n  microns).  There  are  varieties 
from  horse,  cat,  and  bird.  The  lesions  are  more  inflammatory  than  those  of 
the  endothrix  class.  Most  of  the  beard  and  body  ringworms  belong  to  this 
group — very  few  scalp  cases.  The  cultures  are  finely  rayed. 


122 


STUDY   AND    IDENTIFICATION   OF   MOULDS 


Some  give  yellow  cultures,  others  white  and  one  derived  from  birds  a  rose- 
colored  culture. 

Microsporum  audouini. — This  is  the  so-called  small-spored  ring- 
worm and  is  a  very  common  and  highly  contagious  affection  of  the  scalp 
in  children  in  England  and  France;  less  so  in  other  countries. 

It  is  almost  never  seen  in  the  tropics.  It  almost  exclusively  affects  the  hairy 
scalp.  The  spores  are  2  to  3^  in  diameter.  The  broken  stump  of  the  hair  is  char- 
acteristic. The  fungus  is  packed  as  a  mosaic  of  spores,  forming  a  white  sheath, 
chiefly  on  the  outside  of  the  hairs.  It  gives  a  downy-white  culture. 


9-08  . 


FIG.  42. — Tropical  fungi,  i,  concentric  rings  of  Aspergillus  concentricus; 
2,  sporangium  of  A.  concentricus;  3,  Aspergillus  pictor;  4,  Microsporoides  minu- 
tissimus;  5,  Trichosporum  giganteum;  6,  black  granules  of  Madurella  mycetomi; 
7,  yellow  grains  of  Discomyces  madurae. 

Achorion  schoenleini  is  the  cause  of  favus.  The  cultures  are  rather 
wrinkled.  It  is  characterized  by  the  scutulum  or  favus  cup.  This  is  a 
sulphur-yellow  pea-sized  cup  with  a  central  lusterless  hair.  Affected 
hairs  may  not  show  a  cup.  Favus  is  not  so  contagious  as  ringworm. 
It  chiefly  affects  the  hairy  scalp,  but  may  also  invade  the  nails  and  even 
the  body. 

Microscopical  examination  shows  great  irregularity  of  spores  and  mycelium,  the 
latter  being  irregularly  disposed  and  of  varying  thickness  and  length  and  wavy 


ACTINOMYCOSIS  123 

instead  of  straight  as  in  Trichophyton.  There  is  also  the  greatest  irregularity  in 
the  refractile  favus  spores — they  are  gnarled  and  bizarre  shaped,  in  contrast  to  the 
regular  ovals  or  spheres  of  the  ringworm  fungus.  Cultures  show  ridges  or 
convolutions. 

In  the  suborder  Carpoascus  we  have  to  consider  the  family  Perisporiaceae.  In 
this  family  the  asci  are  completely  inclosed  by  the  investing  membrane,  the  peri- 
thecium.  When  this  rots  the  spores  are  set  free.  There  are  two  genera  of  interest, 
Penicillium  and  Aspergillus. 

In  Penicillium  we  have  vertical  branches  with  strings  of  conidia.  In  Aspergillus 
these  conidia  arise  from  a  globular  termination  of  the  hypha. 

Penicillium. — While  Penicillium  does  at  times  form  perithecia,  yet  they  char- 
acteristically show  chains  of  spores.     The  common  P.  glaucum  resembles  a  hand 
with  terminal  beads,  more  than  the  hair  pencil,  from  which  the  name  is  derived. 
P.  crustaceum. — Is  the  common  blue-green  mould.     It  has  been  deemed  patho- 
genic in  cases  of  chronic  catarrh  of  the  eustachian  tube  and  in  gastric  hyper- 
acidity. 

Aspergillus. — These  have  sterigmata  carrying  chains  of  spores,  these  sterigmata 
being  little  processes  projecting  out  from  the  knob-like  termination  of  the  aerial 
hypha  (columella).  Of  the  pathogenic  Aspergilli  we  have: 

1.  A.  fumigatus. — This  has  been  considered  as  the  cause  of  pellagra.     A  pul- 
monary mycosis  resembling  phthisis  may  be  due  to  this  species. 

2.  A.  repens. — This  has  been  found  in  the  auditory  canal  and  may  produce  a 
false  membrane. 

3.  A.  flavus. — This  has  been  found  in  the  discharges  of  chronic  ear  diseases. 

4.  A.  nidulans  has  been  reported  as  one  of  the  causes  of  mycetoma  showing  white 
granules.     It  has  also  been  considered  a  cause  of  aural  mycosis. 

5.  A.  concentricus. — This  is  the  cause  of  an  important  tropical  ringworm,  tinea 
imbricata.     The  scales  are  dry,  like  pieces  of  tissue-paper.     There  are  generally 
about  four  rings  which  do  not  heal  in  the  center.     General  appearance  is  that 
of  watered  silk.     There  are  no  inflammatory  lesions.     Common  in  Malay 
peninsula.     Also  found  in  some  parts  of  the  Philippines  and  in  China.     Some 
authorities  consider  the  fungus  to  be  a  Trichophyton. 

6.  A.  pictor. — This  is  the  cause  of  a  skin  affection  of  Central  America.     In  the 
affection  colored  spots  appear  on  the  skin,  chiefly  on  face,  forearms,  and  chest. 
The  disease  is  attended  with  a  mangy  odor.     Spots  are  of  various  colors;  if 
the  superficial  epithelium  is  affected  we  have  a  dark  violet  color.     Deeper 
involvement  gives  red  spots. 

Hyphomycetes. — In  this  order  are  grouped  certain  genera  which 
cannot  properly  be  assigned  to  any  of  the  other  orders.  They  are  also 
designated  Fungi  Imperfecti,  for  the  reason  that  the  fruiting  bodies 
characteristic  of  the  other  orders  have  not  been  satisfactorily  observed. 

Discomyces  bovis. — This  is  the  well-known  ray  fungus,  the  cajise  of  actinomycosis. 
In  man  it  is  at  times  found  in  chronic  suppurative  conditions  attended  with  much 
granulation  tissue.  Such  pus  may  show  small  yellow-gray  granules  about  the 
size  of  a  pin's  head.  When  spread  out  between  two  slides  the  central  portion 


124  STUDY   AND    IDENTIFICATION    OF    MOULDS 

shows  a  network  of  mycelium  with  bulbous  thread-like  rays  going  to  the  periphery. 
The  "clubs "  at  the  periphery  are  degenerate  structures  and  do  not  stain  by  Gram. 
The  central  mycelium  is  Gram  positive.  This  mould  is  essentially  an  anaerobe 
and  should  be  cultivated  in  a  deep  glucose  agar  stab.  It  may  also  be  cultivated 
in  bouillon.  In  this  it  grows  at  bottom.  Growth  is  dry  and  chalky.  In  diagnosis 
look  for  the  little  granules.  Curetting  of  the  sinuses  may  give  the  "ray  fungus" 
when  they  are  not  found  free  in  the  pus. 

Discomyces  madurae. — This  is  a  ray  fungus  found  in  the  yellow  "fish-roe"  granules 
of  madura  foot.  It  is  strictly  aerobic  in  cultures,  thus  differing  from  actinomy- 
cosis.  For  diagnosis  proceed  as  for  D.  bo  vis. 

Madurella  mycetomi. — This  is  the  cause  of  the  black  "gunpowder"  granules  of 
madura  foot.  It  is  a  mycelial  mass  with  rather  oval  shaped  swollen  segments. 
It  is  at  times  cultivable  on  potato  and  agar  as  felted  masses  of  gray  growth,  which 
later  become  almost  black. 

Malassezia  furfur. — This  is  the  fungus  of  tinea  versicolor.  It  is  common  both  in 
temperate  and  in  tropical  climates.  It  is  characterized  by  dirty  yellow  spots 
about  covered  parts  of  the  body.  Scrapings  show  a  profusion  of  mycelial  threads 
and  interspersed  spores.  It  is  very  difficult  to  cultivate.  The  organism  usually 
termed  the  bottle  bacillus  is  really  a  fungus  having  the  characteristics  of  the 
genus  Malassezia.  It  is  thought  to  be  the  cause  of  pityriasis  of  the  scalp. 
Microsporoides  minutissimus. — This  is  generally  considered  as  the  cause  of  Ery- 
thrasma  or  dhobie  itch,  a  very  common  intertrigo  of  the  tropics.  It  is  character- 
jzed  by  its  narrow  mycelium  and  small  spores.  Various  fungi  are  found  in  this 
affection.  Castellani  considers  the  chief  cause  of  dhobie  itch  to  be  a  trichophyton, 
T.  cruris. 

Clinically  this  affection  shows  festooned  areas  of  a  bright  red  color  which  tend 
to  clear  up  in  the  center  becoming  fawn  color.  As  a  result  of  the  intolerable  itching 
and  scratching  the  affection  tends  to  spread  from  its  favorite  sites — the  inner  sur- 
faces of  the  thighs  and  the  scrotum.  The  spores  and  mycelium  are  abundant  at 
the  onset  but  later,  one  may  not  find  any  evidence  of  the  mould.  In  some  of  the 
rapidly  spreading  cases  I  have  found  a  symbiosis  of  fungus  and  coccus,  the  bacterial 
elements  lying  packed  in  aggregations  scattered  through  the  mycelial  ground  work. 

Culturally  these  cocci  were  S.  pyogenes  aureus. 

Trichosporum  giganteum. — This  is  the  cause  of  a  disease  of  the  hairs,  known  in 
Columbia  as  "Piedra,"  so  called  from  the  small  gritty-like  masses  along  the 
length  of  the  hair.  These  spores  are  arranged  like  mosaics  about  the  hair. 
Sporotrichum  beurmanni. — This  fungus  has  a  narrow  mycelium  (2/0  and  branches 
in  all  directions.  The  spores  appear  as  little  grape-like  clusters  of  oval  spores 
(3  to  5ju)  at  the  end  of  a  filament.  It  is  readily  cultivated,  showing  as  a  small 
white  growth  about  the  eighth  day. 

The  fungus  of  Sporotrichosis  develops  in  tissue  by  budding,  not  showing  the 
mycelial  growth  seen  in  artificial  cultures.  Potato  makes  a  good  medium  and  often 
such  cultures  show  pigmentation. 

This  mould  produces  indolent,  glistening,  subcutaneous  tumors  which  are  pain- 
less. They  may  ulcerate  and  give  off  a  brownish  discharge.  They  resemble  tuber- 
culous or  syphilitic  lesions. 

Certain  organisms  which  resemble  both  moulds  and  bacteria,  having  branching 


FUNGI  125 

filamentous  forms  and  at  the  same  time  having  a  spore-like  method  of  reproduction, 
are  known  under  the  names  Streptothrix  or  better  Nocardia.  It  is  chiefly  in  various 
pathological  processes  of  the  lungs  that  they  have  been  observed,  but  in  addition 
they  have  been  noted  in  brain,  glands,  kidney  and  subcutaneous  tissue. 

The  infections  are  most  likely  to  be  confused  with  phthisis  and  glanders.  The 
organisms  are  easily  cultivated  and  in  staining  reactions  are  midway  between  T.  B 
and  Actinomycosis. 

DIAGNOSIS  OF  FUNGI. 

The  most  expeditious  way  to  examine  for  fungi  is  to  treat  the  scales 
or  hairs  with  a  10%  solution  of  caustic  potash  or  soda.  Then  crush 
between  two  slides;  heat  moderately  over  the  flame  and  examine. 

Tribondeau's  method  is  to  treat  the  scales  with  ether,  then  with  alcohol,  and 
finally  with  water.  Next  put  the  sediment  (it  is  convenient  to  use  a  centrifuge) 
in  a  drop  of  caustic  soda  solution.  Cover  with  a  cover-glass,  and  after  the  prepara- 
tion has  stood  about  an  hour  run  glycerine  under  the  cover-glass. 

A  very  satisfactory  method  is  to  scrape  the  scales  with  a  small  scalpel,  and  smear 
out  the  material  so  obtained  in  a  loopful  of  white  of  egg  or  blood-serum  on  a  glass 
slide.  By  scraping  vigorously  the  serum  may  be  obtained  from  the  patient.  After 
the  smear  has  dried,  treat  it  with  alcohol  and  ether  to  get  rid  of  the  fat.  It  may  then 
be  stained  with  Wright's  stain  or  by  Gram's  method.  The  ordinary  Gram  method 
may  be  used  or  the  decolorizing  may  be  done  with  aniline  oil,  observing  the  decolor- 
ization  under  the  low  power  of  the  microscope. 

Yeasts  are  best  examined  in  hanging  drop  on  the  plain  slide  with  vaselined  cell, 
as  given  under  Blood. 

An  excellent  way  to  examine  moulds  is  to  seize  some  of  the  projecting  sporangia 
from  the  surface  of  a  plate  with  forceps  and  mount  in  liquid  petrolatum.  I  have 
found  that  moulds  in  scales  from  skin  or  infecting  various  mites  or  insects  will  show 
a  growth  in  this  medium  when  mounted  on  a  slide  and  covered  with  a  cover-glass. 

CULTIVATION  OF  FUNGI. 

Moulds  grow  well  on  media  with  an  acid  reaction,  so  that  by  adjust- 
ing the  reaction  to  +  2%  or  even  higher,  we  permit  of  the  growth  of  the 
fungi,  but  inhibit  bacterial  development. 

Glycerine  agar,  bread  paste,  or  potato  media  are  all  suitable,  but  the  best  medium 
is  that  of  Sabouraud: 

Maltose,  4      grams. 

Peptone,  i      gram. 

Agar,  i .  5  grams. 

Water,  100      c.c. 

Make  the  reaction  about  +2. 


126  STUDY   AND    IDENTIFICATION   OF   MOULDS 

Before  inoculating  media  with  moulds,  some  recommend  placing  the  material  in 
60%  alcohol  for  one  or  two  hours  to  kill  the  bacteria.  The  moulds  withstand  such 
treatment. 

In  cultivating  moulds  it  is  best  to  use  small  Erlenmeyer  flasks,  containing  about 
one-fourth  of  an  inch  of  media  on  the  bottom,  for  the  development  of  the  colonies. 
In  order  to  separate  the  mould  we  may  take  the  hair  or  scales  on  a  sterile  slide  and 
cut  them  into  small  fragments  with  a  sterile  knife.  Then  moisten  a  platinum  loop 
from  the  surface  of  an  agar  slant,  touch  a  fragment  with  the  loop,  and  when  it  adheres 
transfer  it  to  the  agar  slant.  Make  four  or  five  inoculations  on  the  surface  and 
from  suitable  growth,  after  four  to  seven  days,  inoculate  the  medium  in  the  Erlen- 
meyer flask. 

Plauth  recommends  receiving  the  mould  material  between  two  sterile  glass  slides. 
Seal  the  edge  of  the  slide  with  wax  and  place  the  preparation  in  a  moist  chamber 
for  four  to  seven  days.  From  developing  fungus  growth  inoculate  the  medium  in 
the  Erlenmeyer  flask.  A  Petri  dish  containing  several  layers  of  thoroughly  mois- 
tened filter-paper  in  top  and  bottom  makes  a  satisfactory  moist  chamber. 


CHAPTER  XI. 
BACTERIOLOGY  OF  WATER,  AIR,  MILK,  ETC. 

BACTERIOLOGICAL  EXAMINATION  OF  WATER. 

WHILE  in  a  chemical  examination  as  to  the  character  of  a  water 
there  are  certain  relations  between  the  free  and  albuminoid  ammonias, 
nitrates,  nitrites,  chlorides,  etc.,  which  indicate  the  probable  animal  as 
against  vegetable  nature  of  the  organic  matter  present,  yet  it  is  a  more 
or  less  presumptive  evidence.  In  a  bacteriological  examination  of 
water  the  finding  of  the  colon  bacillus  may  from  a  practical  standpoint 
be  considered  as  positive  evidence  of  human  faecal  contamination. 
Theoretically,  the  possibility  of  organisms  being  present  corresponding 
culturally  to  B.  coli  and  derived  from  cereals  is  to  be  considered.  Also 
the  faeces  of  animals  contain  an  organism  which  cannot  be  differentiated 
from  the  colon  bacillus. 

In  detecting  sewage  contamination  in  water  to  which  varying  amounts  of  sewage 
had  been  added,  it  was  found  that  the  bacterial  tests  were  from  ten  to  one  hundred 
times  more  delicate  than  the  chemical  ones. 

As  showing  sewage  contamination  of  water,  the  presence  of  the  B.  coli  has  been 
generally  accepted  as  the  most  satisfactory  indication.  The  English  authorities 
consider  sewage  streptococci  and  the  spore-bearing  B.  enteritidis  sporogenes  as  of 
value  as  indicators  as  well  as  the  B.  coli — the  presence  of  sewage  streptococci  indi- 
cating very  recent  sewage  contamination  and  that  of  the  B.  enteritidis  sporogenes, 
in  the  absence  of  streptococci  and  colon  bacilli,  as  evidence  of  sewage  contamination 
at  some  period  more  or  less  remote. 

In  the  United  States  the  colon  bacillus  alone  is  considered  the  indi- 
cator of  sewage  contamination,  and  all  tests,  presumptive  or  positive, 
are  based  on  the  presence  of  this  organism. 

It  is  not  the  finding  of  the  colon  bacillus  but  rather  the  question  of 
it's  relative  abundance  that  is  involved  in  a  water  analysis.  Thus  the 
finding  of  one  colon  bacillus  in  50  c.c.  of  water  would  not  have  weight  as 
showing  contamination,  but  the  presence  rather  constantly  of  the  colon 
bacillus  in  i  c.c.  or  less  makes  contamination  of  a  water  supply 
probable. 

127 


T28  BACTERIOLOGY   OF    WATER,    AIR,    MILK,    ETC. 

In  collecting  samples  of  water  for  bacteriological  examination,  the  following 
points  should  be  considered: 

1.  The  bottles,  which  should  have  a  capacity  of  from  25  to  100  c.c.,  should  be 
sterile.     Sterilization  may  be  effected  by  heat  or  by  rinsing  with  a  little  sulphuric 
acid  and  subsequently  washing  out  thoroughly  with  the  suspected  water  before  col- 
lection.    The  utmost  care  must  be  exercised  that  the  fingers  do  not  come  in  contact 
with  the  glass  stopper  of  the  neck  of  the  bottle  while  filling  it.     If  the  specimen  is 
to  be  sent  some  distance,  it  should  be  packed  in  ice  to  prevent  bacterial  development. 
Frankland  states  that  a  count -of  1000  became  6000  in  six  hours  and  48,000  in  forty- 
eight  hours.     In  water  packed  in  ice  for  a  considerable  time,  however,  the  bacterial 
count  may  diminish. 

2.  If  collecting  from  city  water  supplies,  secure  the  sample  direct  from  the  mains 
and  let  the  water  run  from  the  tap  a  few  minutes  before  collection.     If  the  water  be 
taken  from  a  pond,  stream,  or  cistern,  be  sure  that  the  specimen  comes  from  at  least 
10  inches  below  the  surface.     As  sedimentation  is  the  most  important  method  in 
self-purification  of  rivers  and  ponds,  it  will  be  understood  that  any  stirring  up  of  the 
mud  on  the  bottom  will  enormously  increase  a  bacterial  count. 

Quantitative  Bacteriological  Examination. 

if  Deliver  definite  quantities  of  the  water  to  be  examined  into  tubes  of  liquefied 
gelatin  or  agar  and  plate  out  the  same  in  a  series  of  Petri  dishes. 

A  more  practical  method  is  to  deliver  the  water  from  the  graduated  pipette  into 
the  empty  sterile  dish.  The  water  should  be  deposited  in  the  center  of  the  plate 
and  the  melted  gelatin  or  agar  poured  directly  on  the  water  and  then,  carefully 
tilting  to  and  fro,  mix  the  water  and  the  media.  One  set  of  plates  should  be  of  gelatin 
and  incubated  at  room  temperature;  a  similar  set  should  be  of  lactose  litmus  agar  and 
incubated  at  38°  C.  If  the  water  is  highly  contaminated,  it  is  necessary  to  dilute 
it;  thus,  with  river  water,  which  may  contain  from  2000  to  10,000  bacteria  per  c.c., 
a  dilution  of  i  to  100  would  be  desirable. 

Ordinarily  it  will  be  sufficient  to  deliver  from  a  sterile  graduated  pipette  0.2,  0.3, 
and  0.5  c.c.  of  the  water  in  each  of  two  sets  of  plates:  one  set  for  gelatin,  the  other  for 
agar. 

When  gelatin  is  not  at  hand  or  convenient  to  work  with,  the  gelatin  plates  may 
be  replaced  by  others  of  lactose  litmus  agar  for  incubation  at  room  temperature. 
After  twenty-four  hours  at  38°  C.  or  forty-eight  hours  at  20°  C.,  the  count  should  be 
made. 

Example. — Forty  colonies  were  counted  on  the  gelatin  plate  containing  0.2  c.c. 
(1/5)  of  the  water.  The  number  of  organisms  would  be  200  per  c.c.  Ten  colonies 
were  counted  on  the  agar  plate  containing  0.2  c.c.  and  incubated  at  38°  C.  Number 
of  bacteria  developing  at  body  temperature  equals  50  per  c.c. 

There  is  no  strict  standard  as  to  the  number  of  bacteria  a  water  should  contain 
per  c.c.  Koch's  standard  of  100  colonies  per  c.c.  is  generally  given.  It  is  by  the 
qualitative  rather  than  the  quantitative  analysis  that  one  should  judge  a  water. 

If  there  should  be  very  many  colonies  on  a  plate,  the  surface  can  be  marked  off 
into  segments  with  a  blue  pencil.  If  very  numerous,  cut  out  of  a  piece  of  paper  a 
space  equal  to  i  square  centimeter.  By  counting  the  number  of  colonies  inclosed 


COLON  BACILLUS   IN   WATER 


I2Q 


in  this  space  at  different  parts  of  the  plate,  we  can  strike  an  average  for  each  space  of 
i  square  centimeter.  To  find  the  number  of  such  spaces  contained  in  the  plate, 
multiply  the  square  of  the  radius  of  the  plate  by  3.1416.  Then  multiply  this  number 
by  the  average  per  square  centimeter,  and  we  have  the  total  number  of  colonies 
on  the  plate.  This  is  the  principle  of  the  Jeffers  disc. 

The  relative  proportion  between  the  bacterial  count  at  20°  C.  and  that  at  38°  C. 
is  of  great  importance  from  a  qualitative  standpoint,  as  will  be  seen  later. 

2.  Deliver  into  a  series  of  Durham  fermentation  tubes  containing  glucose 
bouillon  and  into  another  series  containing  lactose  bouillon  varying  definite  amounts 
of  the  water  to  be  examined.  In  tubes  showing  the  presence  of  gas  in  both  glucose 
and  lactose  bouillon  the  evidence  is  presumptive  that  the  colon  bacillus  is  present. 
For  the  positive  demonstration  plates  must  be  made  from  such  tubes  as  show  gas. 

It  is  sufficient  to  deliver  from  graduated  pipettes  in  each  series  quantities  of 
water  varying  in  amount  from  o.i  c.c.  to  10  c.c.  In  our  laboratory  we  inoculate 
with  o.i  c.c.,  0.2  c.c.,  0.5  c.c.,  i  c.c.  and  10  c.c.  of  the  suspected  water.  If  the  o.i  c.c. 
tubes  show  gas,  we  have  reason  to  assume  that  the  water  contained  at  least  10  colon 
bacilli  per  c.c.  If  only  the  10  c.c.  tubes  showed  gas — those  with  less  amounts  not 
having  gas — we  would  be  in  a  position  to  state  that  the  water  contained  the  colon 
bacillus  in  quantities  of  10  c.c.,  but  not  in  quantities  of  i  c.c.  or  less.  Many  authori- 
ties regard  water  as  suspicious  only  when  the  colon  bacillus  is  present  in  quantities 
of  10  c.c.  or  less;  waters  of  good  quality  frequently  showing  the  presence  of  the  colon 
bacillus  in  quantities  of  ico  to  500  c.c. 

It  is  generally  accepted  that  if  a  water  shows  the  presence  of  the 
colon  bacillus  in  quantities  of  i  c.c.  or  less,  it  should  be  regarded  as 
suspicious. 

At  the  present  time  the  medium  that  gives  the  least  source  of  error  in  carrying 
out  the  quantitative  presumptive  tests  is  the  lactose  bile.  It  is  made  by  adding 
i%  of  lactose  and  i%  of  peptone  to  ox  bile,  and  fermentation  tubes  of  the  media 
showing  gas  may  be  considered  as  very  probably  containing  the  colon  bacillus.  The 
percentage  of  error  with  this  method  is  reported  to  be  only  11%,  while  with  glucose 
fermentation  tubes  the  error  is  more  than  50%.  Gas  formation  is  usually  shown  in 
forty-eight  hours,  but  it  is  advisable  to  continue  the  incubation  for  seventy-two  hours. 
These  presumptive  tests  are  chiefly  of  value  in  highly  contaminated  waters.  Even 
with  this  method  plates  should  be  made. 

3.  As  the  colon  and  sewage  streptococci  ferment  lactose  with  the  production 
of  acid  and  hence  produce  pink  colonies  on  lactose  litmus  agar,  much  information 
can  be  obtained  from  the  proportion  existing  between  the  number  of  pink  colonies 
and  those  not  having  such  a  color.  Waters  of  fair  degree  of  purity  rarely  give  any 
pink  colonies. 

Qualitative  Bacteriological  Examination. 

General    Considerations. — In    some    countries    the   proportion  of 
liquefying  to  nonliquefying  colonies  on  gelatin  plates  is  considered  of 
importance.     Certain  sewage  organisms  belonging  to  the  proteus  and 
9 


130  BACTERIOLOGY   OF    WATER,    AIR,    MILK,    ETC. 

cloaca  groups  liquefy  gelatin;  consequently,  if  the  proportion  of  liquefy- 
ing to  nonliquefying  be  greater  than  as  i  to  10,  the  water  is  considered 
suspicious.  The  test  is  not  considered  by  American  authorities  as  of 
any  particular  value. 

The  American  Public  Health  Association  recognizes  the  importance 
of  the  information  obtained  from  a  comparison  of  the  number  of  organ- 
isms developing  at  38°  C.  and  those  developing  at  20°  C.  Bacteria 
whose  normal  habitat  is  the  intestinal  canal  naturally  develop  well  at 
body  temperature,  while  normal  water  bacteria  prefer  the  average 
temperature  of  the  water  in  rivers  and  lakes.  Consequently  when  the 
number  of  organisms  developing  at  38°  C.  at  all  approximates  the  num- 
ber developing  at  20°  C.,  there  is  a  strong  suspicion  that  sewage  or- 
ganisms may  be  present.  Normal  waters  give  proportions  of  i  to  25 
or  i  to  50,  while  in  sewage  contaminated  waters  the  proportion  may  be 
as  i  to  4  or  less. 

In  addition,  the  appearance  of  pink  colonies  on  the  lactose  litmus 
agar  is  of  great  assistance  in  judging  of  a  water.  Both  sewage  strepto- 
cocci and  the  colon  bacillus  give  pink  colonies — 'those  of  the  streptococci 
are  smaller  and  more  vermilion  in  color.  Microscopic  examination 
will  differentiate  the  cocci  from  the  bacilli.  It  is  well  to  bear  in  mind 
that  the  pink  colonies  after  twenty-four  hours  may  turn  blue  in  forty- 
eight  hours  from  the  development  of  ammonia  and  amines.  Conse- 
quently the  lactose  litmus  agar  plates  should  be  studied  after  twenty- 
four  hours. 

A  good  water  supply  will  rarely  show  a  pink  colony,  while  in  a  sew- 
age-contaminated one  the  pink  colonies  will  probably  predominate. 

The  diagnostic  characteristics  considered  important  by  the  Ameri-' 
can  authorities  in  reporting  the  colon  bacillus  (Recently  designated 
excretal  colon  bacillus)  are: 

1.  Typical  morphology,  nonsporing  bacillus,  relatively  small  and  often  quite 
thick. 

2.  Motility  in  young  broth  cultures.     (This  is  at  times  unsatisfactory,  as  some 
strains  of  the  colon  bacillus  do  not  show  it  even  in  young  bouillon  cultures). 

3.  Gas  formula  in  dextrose  broth.     Of  about  50%  of  gas  produced,  one- third 
should  be  absorbed  by  a  2%  solution  of  sodium  hydrate  (CC^).     The  remaining  gas 
is  hydrogen.     (Later  views  indicate  that  the  gas  formula  is  exceedingly  variable  and 
should  not  be  depended  upon.     To  carry  out  this  test  one  fills  the  bulb  of  a  fermen- 
tation tube  with  the  caustic  soda  solution,  holding  the  thumb  over  the  opening 
or  witih  a  rubber  stopper,  the  bouillon  culture  and  the  soda  solution  are  mixed 
by  tilting  the  fermentation  tube  to  and  fro.     The  total  amount  of  gas  is  first  re- 


TYPHOID   AND    WATER  131 

corded  and  then  that  remaining  after  the  CO2  has  been  absorbed  is  reported  as 
hydrogen.) 

4.  Nonliquefaction  of  gelatin. 

5.  Fermentation  of  lactose  with  gas  production. 

6.  Indol  production. 

7.  Reduction  of  nitrates  to  nitrites. 

To  these  may  be  added  the  acidifying  and  coagulation  of  litmus  milk  without 
subsequent  digestion  of  the  casein.  The  production  of  gas  and  fluorescence  in  glu- 
cose neutral  red  bouillon  is  also  a  very  constant  function  of  the  colon  bacillus. 
B.  coli  aerogenes  is  similar  to  B.  coli  with  the  exception  of  nonmotility  and  production 
of  gas  in  saccharose  media.  B.  coli  anaerogenes  is  also  similar  to  B.  coli  but  does  not 
produce  gas  in  glucose  and  lactose. 

NOTE. — The  reduction  of  neutral  red  with  a  greenish-yellow  fluorescence  is  very 
striking  and  has  been  suggested  as  a  test  for  the  colon  bacillus.  Many  other  organ- 
isms, especially  those  of  the  hog  cholera  group,  have  this  power.  It  is  convenient, 
however,  to  color  glucose  bouillon  with  about  i%  of  a  1/2%  solution  of  neutral  red. 

On  the  plates  made  for  the  detection  of  colon  bacillus  may  be  found 
certain  organisms  which  have  origin  in  fecal  contamination.  The  more 
important  of  these  are  those  of  the  paratyphoid,  cloaca  and  proteus 
groups.  In  addition,  the  B.  fecalis  alkaligines  has  not  rarely  been 
isolated.  Among  natural  water  bacteria  there  may  be  present  either 
the  liquefying  or  the  nonliquefying  B.  fluorescens.  These  colonies 
have  a  yellowish-green  fluorescence. 

Certain  chromogenic  cocci  and  bacilli  are  found  in  uncontaminated 
waters  as  B.  indicus  or  B.  violaceus.  From  surface  washings  we  obtain 
certain  soil  bacteria  as  B.  mycoides,  B.  subtilis,  B.  megatherium.  One 
of  the  higher  bacteria  which  shows  long  threads,  Cladothrix  dichotoma, 
is  common,  and  is  characterized  by  a  brown  halo  around  it's  gelatin 
plate  colony. 

Isolation  of  the  Typhoid  Bacillus  from  Water. 

This  is  probably  the  most  discouraging  procedure  which  can  be 
taken  up  in  a  laboratory.  Only  the  most  recent  reports  of  such  isola- 
tion from  water  supplies,  which  have  been  verified  by  immunity  reac- 
tions, can  be  accepted  and  of  these  the  number  of  instances  is  exceed- 
ingly small.  Owing  to  the  long  period  of  incubation,  the  typhoid 
organisms  may  have  died  out  before  the  outbreak  of  an  epidemic 
suggests  the  examination  of  the  water  supply. 

There  have  been  various  methods  proposed  for  the  detection  of  the  B.  typhosus 
in  water.  A  method  which  would  offer  about  as  reasonable  a  chance  of  success  as 
any  other  would  be  to  pass  2  or  3  liters  of  the  water  through  a  Berkefeld  filter;  then 


132  BACTERIOLOGY   OF   WATER,    AIR,    MILK,    ETC. 

to  take  up  in  a  small  quantity  of  water  all  the  bacteria  held  back  by  the  filter.  Then 
plate  out  on  lactose  litmus  agar  and  examine  colonies  which  do  not  show  any  pink 
coloration.  The  dysentery  bacillus  has  about  the  same  cultural  characteristics  as 
the  typhoid  one,  so  that  it  is  important  to  note  motility.  If  from  such  a  colony 
you  obtain  an  organism  giving  the  cultural  characteristics  of  B.  typhosus,  carry  out 
agglutination  and  preferably  bacteriolytic  tests  as  well.  Some  strains  of  typhoid, 
especially  when  recently  isolated  from  the  body,  do  not  show  agglutination. 

The  Conradi  Drigalski,  the  malachite-green,  and  various  caffeine  containing 
plating  media  have  been  highly  recommended. 


Isolation  of  the  Cholera  Spirillum  from  Water. 

The  method  proposed  by  Koch  in  1893  does  not  seem  to  have  been  improved 
upon  by  later  investigators.  To  100  c.c.  of  the  suspected  water  add  i%  of  peptone 
and  i%  of  salt.  Incubate  at  38°  C.,  and  at  intervals  of  eight,  twelve,  and  eighteen 
hours  examine  microscopically  loopfuls  taken  from  the  surface  of  the  liquid  in  the 
flask.  So  soon  as  comma-shape  organisms  are  observed,  plate  out  on  agar.  The 
colonies  showing  morphologically  characteristic  organisms  should  be  tested  as  to 
agglutination  and  bacteriolysis.  Inasmuch  as  the  true  cholera  spirillum  shows  a 
marked  cholera-red  reaction  it  is  well  to  inoculate  a  tube  of  peptone  solution  from 
such  a  colony  and  add  a  drop  of  concentrated  sulphuric  acid  after  incubating  for 
eighteen  hours.  The  rose-pink  coloration  is  given  by  the  cholera  spirillum  with  the 
acid  alone — the  nitroso  factor  in  the  reaction  being  produced  by  the  organism. 


BACTERIOLOGICAL  EXAMINATION  OF  MILK. 

A  bacterial  milk  count  is  of  comparatively  little  value  as  showing 
whether  a  milk  is  dangerous  or  not.  As  a  matter  of  fact,  a  milk  which 
contains  several  million  of  bacteria  per  c.c.  might  be  less  dangerous 
than  one  containing  only  a  few  thousand,  especially  if  in  the  latter 
there  were  numerous  liquefiers  and  gas  producers  present.  There  is, 
however,  one  point  of  importance  in  connection  with  the  quantitative 
estimation  of  bacteria  in  milk,  and  that  is  the  fact  that  in  order  to  keep 
the  development  of  the  bacteria  within  the  limits  of  10,000  to  50,000 
per  c.c.,  it  is  necessary  that  the  requirements  of  cleanliness  in  milking 
and  the  rapid  cooling  of  the  milk  after  obtaining  it  and  the  keeping  of 
the  temperature  below  50°  C.  be  rigidly  observed.  If  a  milk  has  a 
high  count  it  shows  some  error  in  the  handling  of  the  milk.  Anderson 
has  found  that  top  milk  contains  from  ten  to  five  hundred  times  as  many 
bacteria  as  bottom  milk.  Centrifugally  raised  cream  contains  more 
bacteria  than  that  forming  by  gravity.  In  making  a  quantitative  bac- 
teriological examination,  the  principle  is  the  same  as  with  water. 


MILK  I33 

Make  a  known  dilution  of  the  milk  with  sterile  water;  add  definite  quantities  of 
this  diluted  milk  to  tubes  of  melted  <.  gar  or  gelatin,  and  pour  into  plates.  The  diluted 
milk  may  also  be  delivered  in  the  center  of  the  plate  and  the  melted  agar  or  gelatin 
poured  directly  on  it,  mixing  thoroughly.  Always  shake  the  bottle  well  before 
taking  sample. 

Example. — Added  i  c.c.  of  milk  to  199  c.c.  of  sterile  water  in  a  large  flask  (500 
to  1000  c.c.)  After  shaking  thoroughly,  take  i  c.c.  of  this  i  :  200  dilution  and  add 
it  to  99  c.c.  of  sterile  water.  Shaking  thoroughly,  we  have  a  dilution  of  1:20,000. 
Of  this  we  added  0.5  c.c.  to  a  tube  of  gelatin  or  agar.  After  incubation  the  plate 
showed  75  colonies.  Therefore  the  milk  continued  in  each  c.c.  75  X  2X  20,000  (dilu- 
tion) =  3,000,000 — the  number  of  bacteria  in  each  c.c.  of  milk. 

Lactose  litmus  gelatin  or  agar  is  to  be  preferred  in  milk-work,  as  the  normal 
lactic  acid  bacteria  produce  reddish  colonies  which  are  very  striking.  A  standard 
easily  attained  for  high-grade,  certified  milk  would  be  5000  to  10,000  per  c.c. 

In  the  qualitative  examination  of  milk,  many  dairies  employ  the  fermentation 
tube,  any  organism  producing  gas  being  considered  undesirable.  Again  liquefying 
organisms,  as  shown  by  the  presence  of  such  bacteria  in  the  gelatin  plates,  is  evidence 
of  probable  contamination  by  faecal  bacteria.  A  question  which  seems  difficult  to 
decide  is  as  to  the  general  nature  of  the  so-called  normal  lactic  acid  bacteria  of  milk. 
Some  describe  them  as  very  short,  broad  bacilli  with  very  small  colonies,  fermenting 
lactose  with  the  formation  of  lactic  acid.  Others  consider  that  the  streptococci  are 
the  organisms  which  are  concerned  with  the  normal  fermentative  changes.  In 
examining  specimens  of  milk  considered  the  best  on  the  market,  I  have  repeatedly 
found  the  small  red  colonies  on  lactose  litmus  agar  to  be  in  chains  of  either  Gram 
positive  streptococci  or  streptobacilli.  Of  the  acid-forming  bacilli  in  milk  we  have 
i.  the  B.  lactis  acidi  group.  These  are  oval  cells  about  0.9  microns  by  0.6  microns, 
often  in  chains.  They  are  Gram  positive  and  nonmotile.  They  may  be  the  same 
as  Streptococcus  lacticus  of  Kruse.  They  curdle  milk  with  a  homogeneous  clot — this 
being  due  to  the  fact  that  they  do  not  produce  gas  in  lactose  media.  2.  The  B. 
coli  aerogenes  group.  These  are  gas  producers.  (See  under  water.)  3.  The  B. 
bulgaricus  group.  In  connection  with  the  organisms  present  in  the  tablets  used 
for  treating  milk  to  produce  lactic  acid  for  the  treatment  of  intestinal  disorders,  and 
considered  to  be  normal  lactic  acid  bacteria,  I  have  found  both  streptococci  and 
bacilli.  These  have  all  agreed,  however,  in  not  producing  gas  in  either  lactose  or 
glucose  fermentation  tubes. 

The  organism  upon  which  special  stress  is  laid  in  these  so-called  lactic  acid  pro- 
ducers is  the  B.  bulgaricus.  This  is  a  large,  nonmotile  organism  with  square  ends 
like  anthrax.  It  often  occurs  in  long  chains  and  does  not  possess  spores.  It  is  Gram 
positive  and  often  shows  metachromatic  granules  like  those  of  the  diphtheria  bacillus. 
Colonies  show  in  forty-eight  hours  which  resemble  streptococcus  ones,  but  are 
more  contoured  on  the  surface.  It  produces  a  deep  vivid  pink  in  litmus  milk,  while 
milk  streptococci  only  cause  a  light  pink.  It  produces  a  very  large  amount  of  acid 
(3%).  Little  or  no  growth  on  ordinary  laboratory  media  or  below  O°  C.  (Op.  temp. 
42°  C.). 

Heinemann  states  that  it  occurs  normally  in  human  faeces  and  various  fermented 
milks — also  in  gastric  juice  when  HC1  is  absent.  To  isolate,  put  milk  or  faeces  into 
a  broth  containing  0.5%  acetic  acid  and  2%  glucose.  Transfer  to  litmus  milk  after 


134  BACTERIOLOGY   OF   WATER,   AIR,    MILK,   ETC. 

twenty-four  hours  and  from  such  tubes  plate  out  on  milk  serum  agar  (coagulate 
boiling  milk  with  a  few  drops  of  acetic  acid,  filter  and  add  i%  peptone,  2%  glucose 
and  1.5%  agar). 

As  they  grow  in  very  acid  media  the  term  acidophilous  is  applied.  It  was  sup- 
posed that  these  bacteria  were  peculiar  to  certain  fermented  milks  as  matzoon  and 
yogurt.  Hastings  has  shown  the  group  to  be  present  in  milk  in  the  United  States 
and  considers  the  source  to  be  the  alimentary  tract  of  cows. 

Another  source  of  information  as  to  the  quality  of  a  milk  may  be 
derived  from  a  study  of  the  number  of  leukocytes  or  pus  cells  contained 
in  i  c.c.  of  the  milk.  It  must  be  understood  that  cellular  elements 
which  differ  only  slightly  from  true  pus  cells  may  be  found  in  the  milk 
of  healthy  cows  and  may  be  found  in  great  numbers.  Statements  have 
been  made  that  such  cells  are  neither  amoeboid  nor  phagocytic. 

The  Doane-Buckley  method  is  probably  the  most  accurate.  In  this  you  throw 
down  the  cellular  contents  of  10  c.c.  of  milk  in  a  centrifuge  revolving  about  1000 
times  a  minute  for  ten  to  twenty  minutes.  Then  remove  supernatant  milk  and  add 
0.5  c.c.  of  Toisson's'  solution  to  the  sediment.  You  thus  have  the  leukocytes  of 
10  c.c.  contained  in  0.5  c.c.  (Concentrated  twenty  times.)  Make  a  haematocytom- 
eter  preparation  as  for  blood  and  find  the  average  munber  of  cells  for  each  square 
millimeter.  Then  multiply  this  by  10  to  get  the  number  of  cells  in  a  cubic  millimeter. 
As  a  cubic  millimeter  is  one  thousand  times  smaller  than  a  cubic  centimeter,  you 
multiply  the  number  per  cubic  millimeter  by  1000.  Then,  as  the  milk  was  concen- 
trated twenty  times,  you  divide  by  20.  (If  it  were  diluted  twenty  times,  you  would 
multiply  by  20.) 

Example. — Found  an  average  of  50  cells  per  square  millimeter.  This  would 
make  500  per  cubic  millimeter,  and  500,000  per  c.c.;  then  500,000  divided  by  20 
would  give  25,000. 

There  is  no  agreement  as  to  a  standard  for  allowable  leukocytes.  Even  in 
apparently  healthy  animals  they  may  exceed  100,000  per  c.c.  Doane  has  suggested 
500,000  per  c.c.  as  a  preferable  limit. 

The  smear  methods  for  determining  the  number  of  leukocytes  present  do  not 
compare  in  accuracy  with  the  volumetric  ones. 

To  summarize,  we  may  state  that  the  bacterial  count  is  an  indicator 
of  the  care  used  in  handling  the  milk  while  the  presence  of  harmful 
bacteria  (qualitative  examination)  or  numerous  pus  cells  indicates 
disease  in  the  cow.  During  1912  severe  epidemics  of  sore  throat  due 
to  a  streptococcus,  S.  epidemicus,  were  traced  to  milk  of  cows  having 
probably  suffered  from  mastitis.  In  Baltimore  the  milk  had  been 
pasteurized  by  the  flash  method  which  indicates  the  unreliability  of  this 
process. 

Pasteurization  of  Milk.— The  objections  to  this  method  of  preserving  milk 
have  been(i)  that  the  lactic  acid  bacteria  which  have  been  by  some  credited  with 


AIR  135 

antagonism  to  harmful  bacteria,  would  be  destroyed  by  pasteurization,  (2)  the  more 
rapid  development  of  bacteria  in  milk  that  has  been  pasteurized  (3)  interference  with 
nutritive  qualities  and  (4)  pasteurized  milk  does  not  show  its  deterioration  as  does 
unpasteurized  milk,  thus  failing  to  give  a  clue  as  to  the  age  of  the  milk. 

The  United  States  Bureau  of  Animal  Industry  in  studying  this  important  phase 
of  the  milk  question  has  grouped  the  milk  bacteria  into  three  classes  (a)  acid -forming, 
(b)  putrefactive  (liquefying)  and  (c)  inert  bacteria.  In  their  investigations  it  was 
found  that  many  acid- forming  bacteria  withstood  temperature  as  high  as  168°  F., 
so  that  pasteurized  milk  was  soured  just  as  is  raw  milk,  but  more  slowly.  They  found 
that  pasteurized  milk  showed  fewer  putrefactive  bacteria  than  raw  milk,  so  that 
even  should  it  be  a  fact  that  injurious  toxins  were  produced  by  spore-bearing  putre- 
factive organisms  the  development  of  such  organisms  would  be  even  less  in  pasteu- 
rized milk. 

The  statement  so  often  advanced  that  bacteria  develop  more  rapidly  in  pasteu- 
rized milk  than  in  raw  milk  was  proved  fallacious. 

It  was  recommended  that  holding  the  milk  for  thirty  minutes  at  145°  F.  was  a 
far  better  method  of  pasteurizing  than  quickly  bringing  the  milk  to  a  temperature  of 
185°  F.  (flash  method).  All  admit  the  great  value  of  the  killing  of  important  patho- 
gens (typhoid,  cholera,  streptococci,  etc.). 

BACTERIOLOGICAL  EXAMINATION  OF  AIR. 

In  Paris  a  cubic  meter  of  air  was  found  to  contain  the  following 
number  of  organisms: 

Suburbs. — 'Winter,  145  moulds,  170  bacteria. 

Summer,  245  moulds,  345  bacteria. 

City  Hall. — -Winter,  1345  moulds,  4305  bacteria. 

Summer,  2500  moulds,  9845  bacteria. 

Air  of  hospitals,  especially  after  sweeping,  may  contain  50,000 
bacteria  per  cubic  meter.  There  does  not  seem  to  be  any  particular 
relation  between  the  amount  of  carbon  dioxide  in  air  and  the  bacterial 
content. 

Petri's  Rough  Method. — Exposure  of  a  lactose  litmus  agar  plate  (capacity  100 
sq.  cm.)  for  five  minutes  will  give  the  number  of  organisms  present  in  ten  liters  of  air. 
Multiply  by  100  for  one  cubic  meter. 

The  two  groups  of  organisms  usually  found  in  air  are  i.  bacteria  and  2.  moulds. 
Moulds  (spores)  may  be  carried  by  currents  of  air;  bacteria,  however,  are  generally 
carried  about  by  particles  of  dust  or  finely  divided  liquids  (spray).  On  the  lactose 
litmus  agar  plate  staphylococci  and  streptococci  show  as  bright  red  colonies. 

Sedgwick-Tucker  Sterile  Granulated  Sugar  Method. — Sterilize  aerobioscope 
and  introduce  granulated  sugar  on  support.  Again  sterilize  (not  over  120°  C.  in 
dry-air  sterilizer).  Allow  a  given  quantity  of  air  to  pass  through;  then  shake  the 
sugar  into  wide  part  of  aerobioscope.  Now  pour  in  10  or  15  c.c.  of  melted  gelatin 


136  BACTERIOLOGY   OF   WATER,   AIR,   MILK,   ETC. 

(40°  C.)  to  dissolve  sugar.  Roll  tubes  as  for  Esmarch  roll  cultures,  and  incubate 
at  room  temperature.  To  draw  air  through  the  aerobioscope,  connect  the  small  end 
with  a  piece  of  rubber  tubing  which  is  attached  to  a  tube  in  the  stopper  of  an  aspirat- 
ing bottle.  Having  poured  a  definite  quantity  of  water  into  the  aspirating  bottle, 
allow  the  water  to  run  out.  The  same  quantity  of  air  will  be  drawn  through  the 
sugar  of  the  aerobioscope  as  the  amount  of  water  passing  out  of  the  aspirating  bottle. 
The  bacteria  and  moulds  are  caught  by  the  sugar. 

Example. — Passed  ten  liters  of  air  through  the  aerobioscope.  The  bacteria  in 
this  quantity  of  air  showed  75  colonies  when  incubated  at  20°  C.  The  unit  being 
one  cubic  meter  or  one  thousand  liters,  we  have  only  obtained  the  bacteria  of  one 
hundredth  of  the  unit.  Hence  multiplying  75  by  100  gives  7500  bacteria  as  present 
in  one  cubic  meter  of  the  air  examined. 


FIG.  43. — Sedgwick-Tucker  aerobioscope.     (Williams.) 

In  comparing  the  results  with  the  aerobiscope  with  those  obtained 
by  exposing  a  plate  as  in  Petri's  method  for  ten  instead  of  five  minutes, 
it  was  found  that  the  latter  was  sufficiently  in  accord  to  make  it  a  satis- 
factory approximate  quantitative  method.  The  simplicity  and  ease  of 
access  to  the  colonies  developing  on  it  make  it  preferable  when  the  air  of 
operating-rooms  or  hospital  wards  is  to  be  examined. 

Of  the  fungi  ordinarily  obtained  in  examinations  of  the  air  the  blue-green  mould 
and  the  red  yeast  are  the  most  common.  B.  subtilis  and  sarcina  types  of  cocci  are 
the  most  common  bacterial  colonies  found  upon  exposed  plates.  Sewer  air  is  as  a 
rule  free  from  bacteria,  due  probably  to  the  fact  that  bacteria  tend  to  adhere  to 
moist  surfaces.'  The  importance  of  Fliigge's  droplet  method  of  contamination  of 
the  air  of  a  room  is  brought  out  in  the  discussion  of  infection  with  pneumonic  plague. 
This  is  an  important  method  in  the  transmission  of  tuberculosis. 


CHAPTER  XII. 
PRACTICAL  METHODS  IN  IMMUNITY. 

THAT  which  prevents  the  gaining  of  a  foothold  by  disease  organisms 
in  the  animal  body  or  which  neutralizes  their  harmful  products  or  de- 
stroys the  parasites  is  termed  immunity.  In  the  main,  the  question 
of  immunity  hinges  on  the  powers  of  resistance  of  the  human  body 
and  the  aggressiveness  or  virulence  of  the  invading  organism.  It  must 
always  be  kept  in  mind  that  immunity  is  only  relative;  thus  the  fowl, 
which  is  practically  immune  to  tetanus,  may  be  made  to  succumb  by 
reducing  its  resistance  by  refrigeration  or  by  increasing  the  amount  of 
poison  introduced.  The  insusceptibility  which  the  fowl  has  to  tetanus 
or  which  man  has  to  many  diseases  of  animals  is  best  termed  inherent 
immunity,  and  is  at  present  only  a  subject  of  theoretical  interest. 
When  immunity  to  a  given  disease  is  obtained  as  a  result  of  an  attack 
of  the  disease  in  question  or  by  laboratory  methods  of  inoculation,  this 
is  termed  properly  an  acquired  immunity,  and  in  the  former  case  is  a 
naturally  acquired  immunity  or  "natural  immunity"  and  in  the  second 
is  an  artificially  acquired  immunity  or  " artificial  immunity." 

Immunity  may  be  divided  into  that  which  is  inherent  and  that  which  is  acquired. 
Inherent  immunity  is  such  as  is  observed  in  the  resistance  of  Algerian  sheep  to 
anthrax  (ordinary  sheep  are  very  susceptible)  or  the  fowl  to  tetanus  and  is  of  interest 
theoretically  rather  than  practically.  Acquired  immunity  may  be  brought  about 
naturally  as  by  an  attack  of  a  disease  or  artificially  by  laboratory  measures. 

As  a  result  of  an  attack  of  a  disease  or  in  response  to  the  stimulus 
of  the  injection  of  the  organisms  or  its  products,  we  have  developed  in 
the  man  so  injected  certain  specific  antagonistic  properties  to  that 
organism,  which  are  usually  demonstrable  in  the  blood-serum  or  other 
body  fluids,  and  to  which  we  apply  the  terms  agglutinating  power, 
opsonic  power,  or  bacteriolytic  power.  The  term  antibody  is  also 
applied.  All  three  powers  may  be  present  together  in  equal  or  in  vary- 
ing degree  or  one  or  more  may  be  absent.  By  agglutinating  power  we 
mean  that  which  causes  evenly  distributed  organisms  to  come  together 
and  form  clumps.  By  opsonic  power  we  mean  that  which  so  alters  the 

137 


PRACTICAL   METHODS   IN   IMMUNITY 


resistance  of  bacteria  that  the  phagocytes  ingest  them.  By  bacterio- 
lytic  power  we  mean  that  which  brings  about  disintegration  or  lysis  of 
the  specific  organism.  The  bacterium  which  causes  the  disease  or 
which  is  used  in  inoculation  for  the  production  of  immunity  is  termed 
the  specific  organism. 

Of  the  different  kinds  of  immunity  only  artificial  immunity  will  be  considered. 
This  may  be  obtained  in  two  ways:  By  injecting  the  bacteria  or  their  products 
into  man  or  animals  and  as  the  result  of  the  activity  of  the  cells  of  the  animal  invaded, 
antibodies  are  formed  which  neutralize  the  toxins  (antitoxins)  or  bring  about  lysis 

of  the  specific  bacteria  (bacterioly- 
sins).  These  antibodies  which  are 
supposed  to  be  thrown  off  (free  re- 
ceptors) from  those  body  cells  which 
have  suitable  fixation  powers  for  the 
invading  toxin  molecule  or  bacterium 
may  remain  potential  for  months  or 
years  and  so  confer  a  more  or  less  en- 
during immunity. 

These  fixation  points  are  known 
as  cell  receptors  and  are  intended  for 
the  assimilation  of  various  foodstuffs 
by  the  cell.  If  destroyed  by  the 
toxin  or  bacterium  they  are  repro- 
duced in  great  excess  by  nature. 

Not  only  may  bacteria  act 
in   this  way  but  foreign  cells, 
such   as   red    cells    or    various 
parenchymatous  cells,  when  in- 
jected, give  rise  to  antagonistic 
substances  which  act  as  factors  in 
their    destruction — haemolysins 
for  red  cells,  cytolysins  for  dif- 
substance  which  is  injected  and 
produced  is  called  an   antigen. 


FIG.  44. — Receptors  of  the  first  order 
uniting  with  toxin.  (Journal  of  the  A  merican 
Medical  Association,  1905,  p.  955.)  a,  Cell 
receptor;  b,  toxin  molecule;  c,  haptophore 
of  the  toxin  molecule;  d,  toxophore  of  the 
toxin  molecule;  e,  haptophore  of  the  cell 
receptor. 

feient  parenchymatous  cells.  The 
in  reaction  to  which  antibodies  are 
This  is  termed  "active  immunity." 


When  we  take  the  serum  of  a  man  or  animal  immunized  actively  and  inject 
it  with  its  contained  antibodies  into  a  second  animal  or  man,  we  confer  an  immunity 
on  the  second  animal;  but  as  his  cells  take  no  active  part  in  the  production  of  the 
immunity,  but  are  only  passive,  we  term  this  immunity  "passive  immunity."  If 
this  serum  which  is  introduced  in  passive  immunity  only  neutralizes  the  toxic  prod- 
ucts of  the  infecting  bacteria,  we  term  it  antitoxic  passive  immunity  and  designate 
the  immune  serum  as  antitoxic  serum.  If  it- destroys  the  organism,  we  call  it  anti- 


TOXINS 


139 


microbic  serum,  and  the  immunity,  antimicrobic  passive  immunity.     Some  immune 
sera  are  both  antitoxic  and  antimicrobic. 

It  is  well  to  remember  that  some  organisms  produce  a  toxin  which  is  given  off 
while  the  bacterium  is  alive;  and  in  other  instances  the  toxin  is  intracellular  and  is 
only  given  off  when  the  bacterium  disintegrates;  consequently,  an  antimicrobic 
serum  may  cause  the  liberation  of  toxin.  Diphtheria,  tetanus,  or  botulism  antisera 
are  instances  of  antitoxic  sera,  while  practically  all  others  are  antimicrobic.  There 
is  but  one  factor  to  consider  in  an  antitoxic  serum  and  that  is  the  protoplasmic 
particles  which  are  thrown  off  from  the  cell  in  response  to  the  injury  incident  to 
the  attack  upon  the  cell  by  the 
toxin  particles.  This  free  particle 
in  the  circulation  represents  the 
entire  mechanism  of  antitoxic- 
immunity.  It  is  capable  of  unit- 
ing with  the  toxin  molecule  and 
neutralizing  its  toxic  power,  or 
rather  so  binding  its  combining 
end  (haptophore  group)  that  it  is 
incapable  of  attaching  itself  to  a 
cell,  so  that  the  poisonous  end  of 
the  toxin  (toxophore  group)  can- 
not have  access  to  the  cell. 

The  term  toxin,  strictly 
speaking,  is  applicable  only 
to  such  bacterial  poisons  as 
(i)  require  a  period  of  in- 
cubation before  being  capa- 
ble of  manifesting  toxic 
symptoms  and  (2)  can  pro- 
duce antitoxins. 


FIG.  45. — Receptors  of  the  second  order 
and  of  some  substance  uniting  with  one  of  them. 
Journal  of  the  American  Medical  Association, 
1905,  p.  1113.)  c,  Cell  receptor  of  the  second 
order;  d,  toxophore  or  zymophore  group  of 
the  receptor;  e,  haptophore  of  the  receptor;  /, 
food  substance  or  product  of  bacterial  disin- 
tegration uniting  with  the  haptophore  of  the 
cell  receptor. 


(For  further  discussion  of  toxins 
and  antitoxins  see  under  diphthe- 
ria, tetanus,  botulism,  and  pyocy- 

aneus  infections.)  In  antimicrobic  sera  we  have  two  factors  to  consider,  the 
first  is  a  protoplasmic  particle  quite  similar  to  the  antitoxin  molecule,  but  which 
in  itself  has  no  power  of  injuring  its  specific  bacterium.  This  particle  is  gen- 
erally referred  to  as  the  amboceptor  or  immune  body.  It  is  the  specific  product  of 
the  activity  of  a  specific  bacterium  or  foreign  cell  against  the  body  cells  attacked. 
It  withstands  a  temperature  above  56°  C.  and  of  itself  is  incapable  of  injuring  the 
bacterium  in  response  to  whose  attack  it  was  produced.  The  second  factor  in  the 
bacteriolysis  of  the  specific  bacterium,  or  the  haemolysis  of  the  specific  foreign  cell, 
is  something  normally  present  in  the  serum  of  every  animal,  and  which  is  capable 
of  disintegrating  a  foreign  cell  or  bacterium,  provided  it  can  have  access  to  the  cell 
or  bacterium  through  an  intermediary  amboceptor  (hence  the  amboceptor  is  some- 


140 


PRACTICAL   METHODS   IN   IMMUNITY 


times  called  an  intermediary  body).  This  something  is  called  the  "complement." 
It  is  by  some  called  "alexine,"  by  others  cytase  (Metchnikoff).  The  complement 
cannot  act  upon  and  destroy  an  invading  bacterium  or  cell  unless  the  amboceptor 
is  present  to  make  the  necessary  connection.  The  complement  is  destroyed  by  a 
temperature  of  56°  C.,  so  that,  if  we  heat  the  serum  from  an  immune  animal  to  56°  C., 
the  complement  it  naturally  contains  is  destroyed,  and  the  amboceptor  it  contains 
which  is  not  injured  by  such  a  temperature,  is  incapable  of  destroying  bacteria  or 
cells,  unless  we  replace  the  complement  which  has  been  destroyed  by  fresh  comple- 
ment. This  is  done  experimentally  by  adding  the  serum  of  a  nonimmunized  animal 
which  contains  the  complement,  but  no  specific  immune  body  (amboceptor)  to  the 
heated  serum.  This  is  termed  "activating,"  and  a  serum  so  treated  is  said  to  be 
"activated."  When  an  immune  serum  has  been  heated  to  56°  C.,  it  is  said  to  have 
been  "inactivated." 


FIG.  46. — Receptor  of  third  order,  and  of  some  substance  uniting  with  one  of 
them.  (Journal  of  the  American  Medical  Association,  1905,  p.  1369.)  c,  Cell  recep- 
tor of  the  third  order — an  amboceptor;  e,  one  of  the  haptophores  of  the  amboceptor, 
with  which  some  food  substance  or  product  of  bacterial  disintegration  (f)  may  unite; 
g,  the  other  haptophore  of  the  amboceptor  with  which  complement  may  unite; 
k,  complement;  h,  the  haptophore;  z,  the  zymotoxic  group  of  complements. 

Antimicrobic  sera  are  not  as  efficient  in  treatment  as  antitoxic  ones. 
It  might  be  that  if  we  could  use  homologous  sera  for  treating  man  instead 
of  the  usual  heterologous  ones  from  the  horse  better  results  might 
obtain. 

It  would  appear  that  a  more  hopeful  outlook  will  obtain  by  combin- 
ing serum  therapy  with  chemo-therapy,  thus  a  combination  of  anti- 
pneumococcic  serum  with  sodium  oleate  seems  capable  of  producing  cura- 
tive results  which  neither  alone  can  bring  about. 

Again,  a  combination  of  vaccination  (active  immunization)  with 


SERUM  DIAGNOSIS  141 

the  injection  of  the  antimicrobic  serum  (passive  immunization)  has  been 
thought  by  some  to  be  of  value.  When  we  allow  a  mixture  of  bac- 
teria or  cells  to  remain  in  contact  with  their  specific  immune  serum 
which  has  been  inactivated,  the  amboceptors  attach  themselves  to  the 
bacteria  or  cells,  so  that  now,  upon  adding  normal  serum  (comple- 
ment), these  bacteria  or  cells  are  so  prepared  that  the  complement 
can  disintegrate  them.  This  experiment  is  termed  "  sensitizing "  and 
cells  so  treated  are  said  to  be  "sensitized." 

METHODS  FOR  OBTAINING  IMMUNE  SERA. 

While  a  convalescent  from  a  disease  may  be  utilized  to  obtain  an 
antitoxic,  agglutinating,  opsonic,  or  bacteriolytic  serum  against  the 
specific  bacterium,  yet  this  is  more  conveniently  obtained  from  an 
animal  which  has  been  immunized  against  the  bacterium  or  cell  in 
question.  The  rabbit  is  the  most  convenient  animal  to  employ  for  the 
production  of  immune  sera  where  the  object  is  to  have  at  hand  a  serum 
for  use  in  diagnosis. 

Where  sera  are  used  on  an  extensive  scale,  as  in  the  production  of 
curative  sera,  larger  animals  are  employed.  There  are  two  applications 
of  serum  diagnosis:  i.  Where  the  bacterium  is  known  and  the  serum 
is  to  be  diagnosed.  2.  Where  the  serum  is  known  and  the  bacterium 
is  to  be  diagnosed. 

The  first  is  employed  by  testing  the  agglutinating  or  bacteriolytic  power  of  the 
serum  taken  from  a  patient  upon  pure  cultures  of  the  organism  which  is  suspected 
as  the  cause  of  the  disease.  The  Widal  test  (agglutination)  is  the  best  instance  of 
this  procedure.  This  method  is  of  practical  value  in  the  diagnosis  only  of  typhoid, 
Maltg,  fever,  and  para-typhoid.  In  diseases  like  cholera  and  bacillary  dysentery, 
the  disease  has  run  its  course  before  agglutinating  power  becomes  apparent  in  the 
serum.  This  method,  however,  may  be  used  to  prove  that  a  convalescent  has  suf- 
fered from  a  suspected  disease.  Thus,  by  testing  the  agglutinating  power  of  a  serum, 
one  or  two  weeks  after  recovery  from  a  suspicious  case  of  ptomaine  poisoning,  we 
may  be  able  to  demonstrate  that  the  case  in  question  was  cholera.  The  second 
method  has  wider  application,  and  is  the  one  in  which  we  use  the  sera  of  animals 
which  have  been  immunized  with  known  bacteria.  Organisms  isolated  from  urine, 
faeces,  or  blood  of  patients,  or  those  obtained  from  water  or  food  supplies  may  be 
identified  by  testing  the  agglutinating,  opsonic,  or  bacteriolytic  power  of  known 
sera  against  them.  This  has  a  wide  range  of  applicability.  The  testing  of  the 
opsonic  power  of  the  sera  in  man  or  animals  immunized  against  plague,  and  possibly 
cerebrospinal  meningitis,  seems  to  give  more  definite  information  than  do  agglutina- 
tion or  bacteriolytic  tests.  With  the  majority  of  other  organisms,  however,  the 
agglutination  test  is  the  one  almost  always  preferred. 


142 


PRACTICAL   METHODS   IN   IMMUNITY 


Even  in  a  small  laboratory  there  are  no  particular  difficulties  in  the  way  of  hav- 
ing on  hand  rabbits  immunized  against  typhoid,  paratyphoid,  Malta  fever,  acid- 
producing  and  nonacid-producing  strains  of  dysentery,  cholera,  etc.  Just  as  we 
inject  men  with  vaccines  prepared  from  various  bacteria  in  opsonic  therapy,  so  we 
inject  animals  to  produce  sera  for  diagnosis.  We  may  use  either  a  bouillon  culture 
or  the  growth  on  agar  slants  taken  up  with  salt  solution  as  the  inoculating  material. 
This  is  heated  for  one  hour  at  60°  C.  to  kill  the  bacteria.  Where  we  desire  to  produce 
a  serum  which  will  disintegrate  red  blood  cells  (haemolytic  serum),  we  inject  about 
5  c.c.  of  the  washed  red  cells  of  the  animal  for  which  we  wish  to  produce  a  specific 


FIG.  47. — i,  Red  cells  -f-  normal  serum.  No  amboceptor.  No  haemolysis.  A, 
Complement;  B,  normal  red  cell.  2,  Red  cells  +  immune  serum.  Complement  and 
amboceptor.  Haemolysis.  C,  Complement;  D,  amboceptor;  E,  haemolyzed  red 
cell.  3,  Red  cells  +  immune  serum  heated  to  56°  C.  Inactivated.  Complement 
destroyed.  No  haemolysis.  F,  Destroyed  complement;  G,  amboceptor;  H,  red 
cells.  4,  Red  cells  +  heated  immune  serum  +  fresh  serum.  (Activated  by  con- 
tained complement.)  Haemolysis.  I,  Destroyed  complement;  J,  fresh  complement; 
K,  amboceptor;  L,  haemolysed  red  cell.  5,  Diagram  showing  antitoxin  production. 
a,  Toxin  molecule;  b,  antitoxin  molecule;  c,  neutralization  of  toxin  by  antitoxin. 
6,  Diagram  showing  bacteriolysin.  d,  Complement;  e,  amboceptor;  /,  bacillus. 


serum.  For  details  see  method  of  preparing  haemolytic  amboceptor  serum  under 
Noguchi's  mcdification  of  Wassermann  test.  For  preparing  a  serum  for  the  bio- 
logical blood  test  we  inject  the  rabbit  with  human  serum  in  quantities  of  about 
5  c.c.  every  fifth  day.  About  one  week  after  the  last  injection  the  antiserum  ob- 
tained from  the  injected  rabbit  should  be  strong  enough  for  one-tenth  of  a  c.c.  to 
produce  turbidity  when  added  to  i  c.c.  of  a  i-iooo  dilution  of  human  serum  in  salt 
solution.  Various  controls  are  necessary  when  used  in  medico-legal  work. 


AGGLUTINATION   TESTS  143 

For  obtaining  an  agglutinating  or  bacteriolytic  serum  for  bacteria  we  inject 
about  i  c.c.  of  the  killed  bacterial  bouillon  culture  subcutaneously  or  into  the  peri- 
toneal cavity  of  the  rabbit.  The  easiest  way  to  inject  the  rabbit  is  to  hold  the  animal 
head  down  and  plunge  the  needle  in  the  median  line  into  the  abdominal  cavity, 
forcing  in  the  contents  of  the  syringe.  The  intestines  gravitate  downward  and  by 
entering  the  needle  below  the  limits  of  the  bladder  we  avoid  injuring  any  vital  part. 
It  may  be  more  satisfactory  to  at  first  inject  only  about  1/2  c.c.,  and  then  if  there  is 
very  little  reaction,  as  shown  by  the  appetite  and  spirits  of  the  rabbit,  to  inject 
about  four  days  later  i  c.c.  About  four  or  five  injections  at  intervals  of  three  to 
five  days  will  usually  produce  an  immune  serum. 

Injection  of  the  antigenic  material  (blood  cells,  serum  or  bacterial  emulsion)  into 
the  marginal  ear  vein  may  be  employed.  With  this  method,  however,  I  have  had 
several  rabbits  die  in  what  was  considered  anaphylactic  shock.  (For  the  method 
of  immunizing  rabbits  to  produce  a  hsemolytic  serum  see  Wassermann  test.)  Some 
animals  do  not  seem  to  be  capable  of  producing  antibodies,  so  that  it  may  be  necessary 
to  use  one  or  more  rabbits  before  a  satisfactory  serum  is  obtained.  The  most  con- 
venient way  of  obtaining  serum  for  a  test  is  to  cut  across  one  of  the  marginal  veins 
of  the  rabbit's  ear,  and  collect  the  blood  in  a  Wright's  U-tube.  Centrifugalizing, 
we  have  the  serum  ready  for  use. 

The  vein  can  be  made  to  stand  out  prominently  by  applying  a  compress  dipped 
into  very  hot  water.  When  a  large  amount  of  serum  is  desired  it  is  better  to  use  a 
test-tube  with  two  pieces  of  glass  tubing  passing  through  a  double  perforated  rub- 
ber stopper.  To  one  of  the  projecting  pieces  of  glass  tubing  a  stout  hypodermic 
needle  is  attached  through  the  medium  of  8  inches  of  rubber  tubing  and  to  the 
second  piece  of  glass  tubing  passing  through  the  stopper  of  the  large  test-tube 
another  piece  of  rubber  tubing  is  attached  for  suction.  To  obtain  blood  from  the 
rabbit  find  the  ensiform  cartilage  and  insert  the  needle  in  the  notch  to  the  left  and 
gently  force  it  upward.  Applying  suction  with  the  mouth  the  blood  flows  into  the 
test-tube  so  soon  as  the  needle  enters  the  heart.  By  placing  the  tube  of  blood  in 
the  refrigerator  the  serum  separates  out  from  the  clot.  The  removal  of  20  to  30  c.c. 
of  blood  does  not  seem  to  affect  the  animals  in  the  least  and  they  can  be  used  in  this 
way  time  and  time  again.  The  immune  body  and  agglutinin  in  serum  remain 
active  for  weeks  when  kept  in  the  refrigerator.  The  complement  and  opsonin, 
however,  begin  to  deteriorate  at  once  and  have  disappeared  by  the  fifth  day.  Con- 
sequently, for  opsonic  and  bacteriolytic  and  haemolytic  experiments,  fresh  serum 
— twelve  to  twenty-four  hours — must  be  used,  or  it  may  be  activated. 


AGGLUTINATION  TESTS. 

There  are  two  methods  of  testing  the  aggultinating  power  of  a 
serum — -the  microscopical  and  the  macroscopical  or  sedimentation 
method. 

i.  For  the  microscopical  method  draw  up  serum  to  the  mark  0.5  of  the  white 
pipette.  Then  draw  up  salt  solution  to  the  mark  n.  This  when  mixed  gives  a 
dilution  of  i  to  20.  One  loopful  of  the  diluted  serum  and  one  loopful  of  a  bouillon 


144  PRACTICAL   METHODS   IN   IMMUNITY 

culture  or  salt  solution  suspension  of  the  organism  to  be  tested  gives  a  dilution  of 
i  to  40.  One  loopful  of  the  1-20  diluted  serum  and  3  loopfuls  of  the  bacterial  sus- 
pension give  a  dilution  of  1-80.  These  two  dilutions  answer  in  ordinary  diagnostic 
tests.  The  red  pipette  with  a  i-ioo  or  1-200  dilution  may  be  used  where  dilutions 
approaching  i-iooo  are  desired.  Having  mixed  the  diluted  serum  and  the  bacterial 
suspension  on  a  cover-glass,  we  invert  it  over  a  vaselined  concave  slide  and  examine 
with  a  high  power,  a  dry  objective  (1/6  in.).  It  is  simpler  to  make  a  ring  of  vaseline 
to  fit  the  cover-glass  and  make  the  mixture  of  diluted  serum  and  culture  in  the  center 
of  this  ring  or  square.  Then  apply  the  cover-glass,  press  it  down  on  the  vaseline 
ring  and  examine  as  with  the  ordinary  hanging  drop.  In  making  dilutions  it  is 
preferable  to  use  salt  solution,  as  the  phenomenon  of  agglutination  requires  the 
presence  of  salts.  Ordinarily,  thirty  minutes  is  a  sufficient  time  to  wait  before 
reporting  the  absence  of  agglutination.  Agglutination  is  more  rapid  at  body 
temperature  than  at  room  temperature.  In  reporting  agglutination,  always  give 
time  and  dilution.  It  is  absolutely  necessary  that  a  control  preparation  be  prepared 
in  every  instance;  that  is,  one  with  the  bacterial  culture  alone  or  with  a  normal 
serum  of  the  same  dilution  as  the  lowest  used.  Some  normal  sera  will  agglutinate 
in  i  to  10  dilution,  and  group  agglutinations  (as  paratyphoid  with  typhoid  serum) 
may  occur  in  i  to  40  or  possibly  higher.  It  is  very  unusual  for  sera  to  agglutinate 
any  other  bacteria  than  the  specific  one  in  dilutions  as  high  as  1-80. 

2.  For  the  macroscopical  or  sedimentation  test,  take  a  series  of  small  test-tubes 
(3/8X3  in.)  and  deposit  i  c.c.  of  salt  solution  in  each  of  the  series.  Now,  having 
taken  an  empty  test-tube,  drop  4  drops  of  serum  in  it  and  then  add  12  drops  of  salt 
solution.  This  approximately  gives  i  c.c.  of  a  1-4  dilution  of  the  serum.  With  a 
rubber-bulb  capillary  pipette,  which  has  been  graduated  to  hold  16  drops  or  i  c.c., 
draw  up  the  contents  of  the  tube  containing  the  i  to  4  serum  and  add  it  to  the  next 
tube  containing  i  c.c.  of  salt  solution.  This  gives  a  dilution  of  i  to  8.  Now  mix 
thoroughly  by  drawing  up  and  forcing  out  with  the  bulb  pipette,  and  then  withdraw 
i  c.c.  and  add  to  the  next  tube  containing  i  c.c.  of  salt  solution.  This  gives  a  dilu- 
tion of  i  to  1 6.  Having  mixed  as  before,  again  withdraw  i  c.c.  of  the  mixture  and 
add  it  to  the  i  c.c.  in  the  next  tube.  We  now  have  a  dilution  of  i  to  32.  Again 
withdrawing  i  c.c.  and  adding  it  to  the  fourth  tube  containing  i  c.c.  of  salt  solution 
we  have  a  dilution  of  i  to  64.  In  tube  i  there  is  i  c.c.  of  a  dilution  of  the  serum  of 
i  to  8;  in  tube  2,  there  is  i  c.c.  of  a  dilution  of  i  to  16;  in  tube  3  of  i  to  32.  Tube 
4  contains  2  c.c.  of  i  to  64.  Now  adding  i  c.c.  of  a  culture  of  typhoid  or  any  other 
organism,  we  have  the  dilution  of  the  serum  in  each  tube  doubled.  Tube  i  now 
contains  a  serum  in  dilution  of  i  to  16,  acting  on  the  bacteria;  tube  2  of  a  i  to  32; 
tube  3  of  a  i  to  64.  Now  place  these  tubes  in  the  incubator  and,  after  two  to  five 
hours  or  overnight,  we  examine  for  the  clearing  up  of  the  supernatant  fluid.  If 
the  serum  in  a  certain  dilution  agglutinates,  the  clumps  gravitate  to  the  bottom 
and  the  upper  part  becomes  clear.  If  so  desired,  these  dilutions  may  be  carried  on 
to  i  to  several  hundred  in  the  same  way.  It  is  safer  to  work  with  dead  cultures 
instead  of  living  ones.  To  prepare,  take  a  twenty-four-hour  agar  slant  culture  of 
typhoid  or  paratyphoid  and  emulsify  in  salt  solution  (about  6  c.c.  to  a  slant). 

By  adding  o.i  of  i%  of  formalin  to  the  typhoid  emulsion  and  placing  in  the  ice- 
box the  cultures  will  be  found  sterile  in  about  three  days.  The  emulsion  should  be 
shrken  twice  daily  while  undergoing  sterilization  in  the  ice-box.  Such  cultures 


COMPLEMENT  DEVIATION  145 

are  not  easily  contaminated  and  appear  to  retain  their  agglutinable  qualities  for 
several  months.     The  macroscopic  methods  are  preferable  with  such  dead  cultures. 

A  very  convenient  method  in  general  use  in  Germany  is  the  follow- 
ing: Make  dilutions  of  serum  in  ordinary  test-tubes  (3/4X6  in.)  as 
described  for  the  samll  test-tubes.  Then  take  a  loopful  (2  mg.)  of 
culture  from  an  eighteen  to  twenty-four-hour-old  agar  culture  and 
emulsify  it  thoroughly  in  the  dilution  in  the  first  test-tube — repeat  the 
process  in  the  second  tube  and  so  on.  This  procedure  is  much  safer 
than  when  live  cultures  are  added  with  a  pipette.  Again,  the  dilution 
is  unchanged  by  this  addition  whereas  it  is  doubled  when  an  equal 
volume  of  culture  is  added  to  the  diluted  serum.  A  control  should 
always  be  made  in  normal  salt  solution.  After  incubating,  observe 
flocculent  precipitates  (agglutination)  by  tilting  the  fluid  in  the  tubes 
to  form  a  thin  layer  and  to  obtain  the  most  advantageous  light  and  look 
for  a  fine  curdy  precipitate  (aggutination)  or  a  uniformly  turbid  emul- 
sion (negative  reaction). 

The  method  of  using  a  slide  with  two  vaselined  rings,  one  containing 
an  emulsion  in  the  specific  serum  and  the  other  in  salt  solution  is  of 
great  practical  value.  This  method  is  described  under  cholera. 

Pfaundler  under  the  designation  of  a  thread  reaction  showed  that  organisms  tended 
to  grow  in  thread  forms  in  a  culture  medium  containing  the  homologous  serum. 
Mandelbaum  has  suggested  this  as  a  means  of  diagnosing  typhoid.  Take  ordinary 
bouillon  containing  1%  of  sodium  citrate.  Inoculate  it  with  a  culture  of  typhoid. 
Now  with  a  bulb  capillary  pipette  take  up  one  part  (as  marked  by  a  wax  pencil)  of 
the  patient's  blood  and  fifteen  times  as  much  of  the  citrated  bouillon  just  inoculated 
with  typhoid.  Mix  the  blood  and  citrated  bouillon  on  a  sterile  slide  or  in  a  test-tube 
and  after  drawing  up  into  the  lower  part  of  the  expansion  of  the  capillary  pipette, 
seal  off  the  capillary  end.  Now  place  the  sealed-off  pipette  upright  in  an  incubator 
and  after  four  or  five  hours  take  out  from  the  expanded  end  a  loopful  of  the  clear 
supernatant  fluid  (the  blood  cells  settle  to  the  bottom)  and  if  the  typhoid  bacilli 
are  in  chains  instead  of  being  single  and  motile  it  shows  a  positive  reaction. 

DEVIATION  OF  THE  COMPLEMENT. 

It  has  been  found  that  if  there  is  not  sufficient  immune  body  in  a 
mixture  of  normal  serum,  containing  abundant  complement,  and  bac- 
terial emulsion,  only  a  portion  of  the  bacteria  will  be  destroyed.  In- 
creasing the  amount  of  immune  body  with  a  constant  quantity  of  normal 
serum,  we  reach  a  point  where  all  the  bacteria  are  destroyed.  Now, 
if  we  continue  to  increase  beyond  this  point  the  addition  of  immune 

10 


146  PRACTICAL   METHODS   IN   IMMUNITY 

serum,  the  destruction  of  the  bacteria  ceases,  and  the  cultures  will  again 
contain  myriads  of  living  bacteria. 

To  carry  out  the  test,  make  a  series  of  tubes  containing  mixtures  of  bacteria 
with  the  same  quantity  in  each  of  normal  serum.  Thus,  each  tube  contains  1/2  c.c. 
of  bacterial  emulsion  and  1/2  c.c.  of  i-io  normal  serum.  Now  inactivate  a  tube  of 
i  -zoo  immune  serum  and  to  each  of  the  tubes  of  normal  serum  and  bacterial  emulsion 
add  increasing  drops  of  the  inactivated  i-ioo  immune  serum.  Thus,  i  drop  to  No.  i 
tube,  2  drops  to  No.  2  tube,  and  so  on.  After  incubating  for  two  hours,  we  take  a 
pipette  and  plate  out  a  fraction  of  a  drop  in  an  agar  plate.  The  limit  at  which  bac- 
teriolysis is  complete  is  shown  by  there  being  an  absence  of  colonies. 

Beyond  or  below  that  point  colonies  are  more  or  less  abundant.  The  explanation 
of  this  phenomenon  of  deviation  or  deflection  of  the  complement  is  that  where  we 
have  an  excess  of  amboceptors  for  available  receptors  on  the  bacterial  cells,  only  a 
portion  of  the  amboceptors  can  attach  themselves  to  their  specific  bacteria.  The 
free  amboceptors,  not  being  able  to  form  a  union  with  the  bacterial  cell  receptors 
(for  which  they  have  a  greater  affinity),  combine  with  the  complement  present. 
Unless  the  complement  be  in  excess,  there  will  be  no  free  complement  left  to  join 
onto  the  amboceptors  attached  to  the  bacterial  cells,  and  consequently  bacteriolysis 
does  not  take  place  and  the  plate  cultures  show  an  abundance  of  colonies. 

FIXATION  OR  ABSORPTION  OF  THE  COMPLEMENT. 

One  of  the  controversies  in  connection  with  the  nature  of  the  com- 
plement is  that  regarding  the  question  of  the  unity  of  complements 
or  whether  there  exist  different  kinds  of  complements  for  different 
amboceptors  (unity  and  multiplicity  of  complement).  To  prove  that 
a  single  complement  will  act  with  varying  amboceptors,  Bordet  and 
Gengou  showed  that  the  same  complement  would  activate  both  haemo- 
lytic  and  bacteriolytic  immune  bodies.  If  to  a  mixture  of  typhoid 
bacteria  and  inactivated  typhoid  immune  serum  some  guinea-pig  serum 
is  added  and  the  mixture  be  allowed  to  remain  at  37°  C.  for  two  hours, 
and  then  sensitized  red  cells  be  added  and  the  mixture  again  be  placed 
in  the  incubator  for  two  hours,  no  haemolysis  will  be  found  to  have 
occurred,  because  the  bacteria  have  absorbed  all  the  guinea-pig  com- 
plement through  the  intervening  typhoid  amboceptors,  and  there  is  no 
complement  left  to  haemolyze  the  red  cells  through  the  specific  blood- 
cell  amboceptors.  If,  instead  of  immune  typhoid  serum,  the  serum  of  a 
.normal  person  had  been  used,  there  would  have  been  no  amboceptors 
to  unite  the.complement  to  the  bacterial  cells.  The  complement  would 
then  be  at  hand  to  unite  with  the  sensitized  red  cells  subsequently  added 
and  bring  about  their  haemolysis,  as  shown  by  the  ruby  color  of  the 


COMPLEMENT   FIXATION  147 

supernatant  fluid.  This  phenomenon  of  Bordet  and  Gengou  has  been 
utilized  by  Wassermann  for  the  diagnosis  of  diseases  where  cultures  are 
not  applicable.  It  is  in  the  diagnosis  of  syphilis  that  it  is  best  known. 
It  having  until  recently  been  impossible  to  obtain  cultures  of  Treponema 
pallidum,  we  use  an  emulsion  of  the  liver  of  a  syphilitic  foetus,  which 
has  been  filtered  so  as  to  be  clear,  instead  of  a  culture.  The  syphilitic 
liver,  as  can  be  observed  by  staining  according  to  Levaditi's  method,  is 
packed  with  spirochaetes. 

While  Noguchi  has  recently  obtained  pure  cultures  of  the  organism  of  syphilis 
yet  the  antigen  prepared  from  such  cultures  was  not  found  as  satisfactory  by  Craig 
and  Nichols  as  that  from  the  liver  of  a  syphilitic  foetus,  cases  of  syphilis  which  showed 
strongly  positive  tests  with  ordinary  antigen  not  giving  a  positive  test  with  the  spe- 
cific antigen. 

It  has  now  been  found  that  lecithin  or,  preferably,  emulsions  of  various  normal 
organs  may  be  substituted  as  antigen  for  the  syphilitic  liver,  the  antigenic  power 
being  due  to  lipoids.  Aqueous  extracts  contain  in  addition  to  lipoids,  substances 
which  render  the  antigen  unstable — alcoholic  extracts  are  more  stable  and  contain 
less  anticomplement. 

For  the  immune  bodies  we  take  the  serum  of  the  patient,  or  if  a  case  of  locomotor 
ataxia  or  general  paresis,  the  cerebrospinal  fluid. 

EMERY'S  TECHNIC  FOR  THE  WASSERMANN  TEST. 

Owing  to  technical  difficulties  with  the  method  of  making  and 
employing  the  antigen  and  amboceptor  features  of  the  original  Emery 
test,  I  have  retained  the  principle  of  the  test  but  substituted  the 
reagents  prepared  in  exact  accordance  with  Noguchi's  directions. 

Briefly  stated,  the  principle  of  Emery's  test  consists  in  the  employ- 
ment of  fresh  human  serum  for  supplying  complement  and  the  pri- 
mary incubating  of  the  hemolytic  system  (human  red  cells  and  rabbit 
serum  immune  to  human  red  cells)  at  the  same  time  as  the  incubation 
of  the  antigen  and  serum  but  in  separate  tubes.  Then  in  the  second 
period  of  incubation  to  add  these  " sensitized"  cells  to  the  serum 
antigen  combination. 

In  Noguchi's  method  all  reagents  are  incubated  together  in  the 
first  period  with  the  exception  of  the  amboceptor  paper  (dried  serum 
of  rabbit  immune  to  human  red  cells),  which  is  not  added  until  the 
period  of  incubation  for  complement  binding  is  completed  and  the 
second  incubation  commenced.  Time  is  saved  in  the  Emery  tech- 
nic,  inasmuch  as  the  red  cells  are  already  sensitized  by  the  hemo- 


148  PRACTICAL   METHODS    IN   IMMUNITY 

lytic  amboceptor  when  added  to  the  tubes,  and  hemolysis  shows  itself 
almost  immediately  in  tubes  when  the  complement  has  not  been 
absorbed  by  the  antigen  through  syphilitic  antibodies. 

Noguchi  has  called  attention  to  the  fact  that  protein  constituents  of  certain 
aqueous  or  alcoholic  extracts  may  have  the  power  to  fix  complement  through  certain 
intermediaries  existing  in  fresh  serum  which,  however,  does  not  obtain  for  inacti- 
vated sera  (sera  heated  to  56°  C.  for  15  minutes). 

Pure  lipoidal  substances  as  contained  in  Noguchi's  acetone  insoluble  antigen, 
however,  do  not  act  in  this  way. 

Consequently  by  using  such  an  antigen  we  eliminate  the  objection  to  employing 
fresh  human  serum  in  the  test  for  syphilitic  antibodies. 

As  giving  more  uniform  hemolytic  results  and  as  being  more  stable  and  easier 
of  employment,  I  have  made  use  of  Noguchi's  directions  for  taking  up  the  serum  of 
the  rabbits,  immunized  to  human  red  cells  and  his  method  of  standardizing  this 
''  amboceptor "  paper.  In  practice,  I  measure  off  the  length  of  paper  correspond- 
ing to  8  to  10  Noguchi  units  and  dissolve  the  dried  serum  in  such  paper  in  i  c.c.  of 
salt  solution.  This  makes  a  satisfactory  and  uniform  substitute  for  the  sterile 
immune  serum  used  by  Emery.  It  has  been  noted  that  the  dried  rabbit  serum  on 
the  paper  may  contain  a  certain  amount  of  complement  even  when  several  months 
old,  consequently,  to  avoid  confusion,  I  invariably  inactivate  this  serum  paper  solu- 
tion by  heating  to  56°  C.  for  5  minutes. 

Method :  i.  Take  blood  from  the  finger  or  ear  in  a  large  Wright  U  tube  (1/4  inch 
in  diameter).  Place  in  37°  C.  incubator  for  15  minutes  (to  increase  yield  of  serum) 
and  then  centrifuge. 

2.  Graduate  a  capillary  pipette  for  i  volume  and  4  volumes. 

3.  Into  each  of  a  series  of  small  test-tubes  put  4  volumes  of  normal  salt  solution. 
(These  tubes  are  most  conveniently  made  by  breaking  off  2  1/2  to  3  inch  lengths  of 
i/4-inch  soft  glass  tubing  and  then  fusing  one  end  in  the  flame  to  make  a  small  test- 
tube.) 

Make  a  distinguishing  mark,  e.  g.,  X,  on  end  of  tube  with  blue- wax  pencil  and  use 
this  tube  to  hold  control.  Mark  the  other  tubes  I,  II,  III,  and  so  on.  When  differ- 
ent sera  are  to  be  tested  they  may  be  distinguished  by  lines  either  above  or  below, 
or  with  circles,  also  marks  with  red-wax  pencil  may  be  used. 

4.  Make  a  i  to  10  dilution  of  stock  antigen  solution  in  salt  solution. 

To  Tube  I  add  4  volumes  of  i  to  10  antigen,  thus  making  8  volumes  of  i  to  20 
antigen  in  Tube  I.  Mix  thoroughly  by  manipulating  bulb  of  pipette.  Then  trans- 
fer 4  volumes  of  the  i  to  20  from  Tube  I  to  Tube  II,  and  so  on  through  the  series. 
When  the  dilution  in  the  last  tube  has  been  made  throw  4  volumes  away. 

The  4  volumes  of  dilution  of  the  antigen  in  the  respective  tubes  will  then  be: 
In  Tube  I,  i  to  20;  in  Tube  II,  i  to  40;  in  III,  i  to  80;  in  IV,  i  to  160,  and  so  on. 

5.  Add  i  volume  of  serum  to  be  tested  to  control  tube  X,  and  to  each  of  the  tubes 
I,  II,  III,  etc.,  in  succession.     (If  the  serum  be  added  to  the  antigen  tubes  before  the 
control  tube,  -antigen  might  be  carried  over  to  the  control.) 

6.  If  the  serum  has  been  inactivated  restore  complement  by  adding  i  volume 
of  a  40%  fresh  guinea-pig  serum.     Also  use  2  volumes  of  this  inactivated  human 
serum  instead  of  i. 


WASSERMANN   TECHNIQUE 


149 


7.  Incubate  at  38°  C.  for  30  minutes.     This  allows  syphilitic  antibody,  if  pres- 
ent, to  bind  complement. 

8.  As  soon  as  the  above  mixtures  have  been  made  and  put  in  the  incubator 
prepare  the  "haemolytic  system"  by  adding  i  volume  of  20%  emulsion  of  washed 
human  red  cells  to  4  volumes  of  solution  of  amboceptor  paper  (8  to  10  Noguchi 
units  of  amboceptor  paper  dissolved  in  i  c.c.  of  salt  solution  and  then  heated  to  55°  C. 
for  5  minutes  makes  a  suitable  amboceptor  solution — thus  of  a  paper  of  which  4  mm. 


CONTROL      1to2°        U'40        Ito8°         I10|6°       't'320      lt,640 
ANTIGEN    ANTIGEN    ANTIGEN    ANTIGEN     ANT1OEN  ANTIGEN 


FIG.  48. — i.  Capillary  pipette  being  graduated  by  drawing  up  i  and  4  drops 
from  a  watch-glass,  (a)  Blue  pencil  mark  of  i  drop  or  i  volume,  (b)  Mark  of 
volume  of  4  drops.  2.  Graduated  centrifuge  tube  containing  sodium  citrate  normal 
salt  solution.  3.  Tube  with  10  amboceptor  units  in  i  c.c.  of  salt  solution.  4.  Mix- 
ture of  i  volume  20%  emulsion  red  cells  and  4  volumes  inactivated  amboceptor 
solution.  5.  Small  glass  tubes  for  Emery  test.  6.  Method  of  transferring  from 
tube  to  tube.  7.  Making  a  Wright  U-tube — the  end  "a"  to  be  used  as  a  capillary 
pipette. 

was  the  unit  we  should  cut  off  about  40  mm.,  place  in  test-tube  and  extract  the  dried 
serum  with  i  c.c.  of  salt  solution),  and  place  this  haemolytic  system  in  incubator 
alongside  the  tubes  already  there.  To  obtain  the  washed  red  cells  allow  4  to  10 
drops  of  blood  to  drop  into  a  graduated  centrifuge  tube  containing  salt  solution  to 
which  has  been  added  i  %  of  sodium  citrate  to  prevent  coagulation.  After  shaking, 
centrifuge.  Pour  off  supernatant  fluid,  replace  with  salt  solution,  again  shake  and 
centrifuge — this  sediment  of  red  cells  is  to  be  diluted  with  4  volumes  of  salt  solution 
(20%  emulsion).  (Incubation  hastens  sensitization  of  the  red  blood  cells.  Agglutina- 
tion of  red  cells  also  occurs.) 


PRACTICAL   METHODS   IN   IMMUNITY 


9.  At  the  expiration  of  30  minutes  from  the  commencement  of  incubation  for 
complement  binding,  add  i  volume  of  haemolytic  system  to  each  of  the  tubes,  I,  II, 
III,  etc.,  in  the  order  of  antigen  dilution. 

10.  Finally,  after  washing  pipette  in  salt  soultion,  add  i  volume  of  haemolytic 
system  to  control  in  tube  X.     (If  the  haemolytic  system  should  be  added  to  the  con- 
trol tube  before  the  antigen  tubes,  complement  from  the  control  tube  might  be 
carried  over  to  the  antigen  tubes.) 


FIG.  49. — i.  Copper  water  bath  12X12X8  inches,  (a)  Thermometer  to  show 
38°  C.  (b)  Tubes  containing  antigen  dilutions,  (c)  Tube  containing  hamolytic 
system  incubating  along  with  the  antigen  tubes.  2.  Ordinary  rice  cooker  with  copper 
holder  for  test-tubes. 

Shake  each  tube  thoroughly.  Allow  them  to  incubate  for  a  few  minutes.  Then 
examine  tubes  I,  II,  III,  etc.,  for  haemolysis.  The  control  should,  of  course,  show 
haemolysis.  The  antigen  tubes  should  show  a  white,  supernatant  fluid  over  the  in- 
tact red  cell  sediment  in  the  tubes  with  the  low  dilutions  and  even  in  the  highest 
dilutions,  where  the  serum  is  strongly  positive.  In  a  weakly  positive  serum,  in- 
hibition of  haemolysis  may  only  show  in  the  first  two  tubes  and  haemolysis  show  in 
those  tubes  having  higher  dilutions  of  antigen. 

It  will  be  noted  that  the  reagents  are  made  in  accordance  with 
Noguchi's  directions.  Even  in  those  cases  where  fresh  guinea-pig 
serum  is  employed  to  replace  complement,  absent  from  the  human 
serum  tested,  we  employ  the  40  %  solutions  used  in  Noguchi's  tech- 


ANTIGEN  151 

nic.  It  is  possibly  better  to  start  with  a  1-30  dilution  in  the  first 
antigen  tube  instead  of  with  a  1-20.  It  is  also  an  advantage  to  titrate 
the  human  complement. 

Preparation  of  Acetone  Insoluble  Antigen. — Take  about  50  grams  of  finely 
divided  beef,  dog,  or  rabbit  heart  or  liver  and  triturate  in  a  mortar  to  a  paste.  Pour 
on  this  paste  500  c.c.  of  absolute  alcohol  and  keep  the  mixture  in  a  corked  bottle 
in  the  37°  C.  incubator  for  five  to  seven  days.  (We  use  beef  heart  and  96%  alcohol.) 
Next  filter  through  paper  and  collect  the  filtrate  in  a  large  shallow  dish  and  hasten 
evaporation  with  the  aid  of  a  current  of  air  from  an  electric  fan  directed  upon  the 
uncovered  surface. 

Within  twenty-four  hours  only  a  sticky  residue  should  remain.  This  is  taken  up 
in  about  50  c.c.  of  ether  and  the  turbid  ethereal  solution  kept  over  night  in  the  refrig- 
erator in  a  corked  bottle. 

In  the  morning  there  will  be  found  about  45  c.c.  of  clear  supernatant  fluid  which 
is  decanted  off  and  allowed  to  evaporate  to  about  15  c.c. 

Now  to  this  15  c.c.  add  about  150  c.c.  of  acetone  and  a  precipitate  will  form 
which  collects  at  the  bottom  of  the  measuring  cylinder.  Now  pour  off  the 
supernatant  acetone  and  let  the  sediment  stand  until  it  is  of  a  resinous  con- 
sistence. Now  dissolve  0.3  grams  in  i  c.c.  of  ether  and  then  add  9  c.c.  of  methyl 
alcohol.  This  gives  the  stock  antigen  solution. 

In  using  the  antigen  solution  for  the  Emery  or  Noguchi  test  we  dilute  i  c.c.  with 
9  c.c.  of  salt  solution.  This  opalescent,  working,  antigen  emulsion  should  be  made 
up  fresh  on  the  day  of  preparing  the  tests. 

About  one-half  of  these  antigens  are  lacking  in  power  to  absorb 
complement  in  the  presence  of  syphilitic  sera.  More  rarely  they 
may  absorb  complement  with  a  nonsyphilitic  serum  (anticomplemen- 
tary)  or  they  may  have  a  haemolytic  action.  Consequently  a  new  stock 
antigen  should  be  tested  as  to  its  reliability— 

1.  A  mixture  of  0.4  c.c.  working  antigen  emulsion,  0.6  c.c.  salt  solution,  and  o.i 
c.c.  of  a  10%  suspension  of  washed  red  cells  when  incubated  at  37°  C.  for  two  hours 
should  not  show  any  haemolysis. 

2.  A  mixture  of  0.4  c.c.  working  antigen  emulsion,  0.6  c.c.  salt  solution,  o.i  c.c. 
of  a  40%  solution  of  fresh  guinea-pig  serum,  and  2  units  of  amboceptor  and  incubated 
at  37°  C.  for  one  hour  should  show  haemolysis  when  we  now  add  o.i  c.c.  of  a  10% 
washed  red-cell  emulsion  and  the  whole  then  again  incubated  for  two  hours  at  37°  C. 
(The  antigen  did  not  absorb  complement  in  the  absence  of  syphilitic  antibodies.) 

3.  A  mixture  of  0.2  c.c.  of  a  i  to  10  dilution  of  working  antigen  emulsion,  0.8 
c.c.  of  salt  solution,  i  drop  of  syphilitic  serum,  o.i  c.c.  of  a  40%  dilution  of  fresh 
guinea-pig  serum,  and  2  units  of  amboceptor  should  be  incubated  at  37°  C.  for  one 
hour.     When  we  then  add  o.i  c.c.  of  a  10%  suspension  of  washed  red  cells  and 
again  incubate  for  two  hours  we  should  fail  to  obtain  haemolysis.    (The  antigen  can 
absorb  complement  through  the  intermediation  of  syphilitic  antibodies.) 

Preparation  of  Amboceptor  Paper. — In  order  to  secure  blood  from  the  vein  of  a 


152  PRACTICAL   METHODS   IN   IMMUNITY 

man  or  the  heart  of  the  immunized  rabbit,  the  most  convenient  method  is  with  the 
use  of  an  Erlenmeyer  flask  with  a  rubber  stopper  having  two  perforations  in  the  stop- 
per. To  one  of  the  projecting  pieces  of  glass  tubing  a  stout  hypodermic  needle  is 
attached  through  the  medium  of  about  8  inches  of  rubber  tubing,  and  the  second 
piece  of  glass  tubing  is  bent  at  an  angle  as  it  leaves  the  stopper  to  provide  a  suction 
tube.  With  a  man,  constrict  the  upper  arm  sufficiently  to  stop  venous  return  with 
an  Esmarck  rubber  bandage  or  a  towel.  Paint  tincture  of  iodine  over  a  prominent 
vein  at  the  bend  of  the  elbow.  Gentle  suction  will  cause  the  blood  to  flow  into  the 
needle  tube  and  thence  into  the  flask. 

The  blood  as  it  is  taken  from  the  arm  should  be  received  in  about  50  c.c.  of  normal 
salt  solution  containing  i%  of  sodium  citrate.  About  28  to  30  c.c.  are  usually  suffi- 
cient. Now  throw  down  this  red-cell  suspension  in  three  or  four  centrifuge  tubes. 
The  resulting  sediment  should  be  washed  and  rewashed  with  salt  solution.  Two 
to  three  washings  with  salt  solution  suffice. 

Now  take  a  large  healthy  rabbit,  shave  the  lower  abdomen  and  paint  the  surface 
with  tincture  of  iodine.  The  easiest  way  to  inject  the  rabbit  is  to  hold  the  animal 
head  down  and  plunge  the  needle  of  a  large  glass  hypodermic  syringe  containing  the 
washed  red-cell  sediment  into  the  abdominal  cavity  in  the  median  line.  The  intes- 
tines gravitate  downward  and  by  entering  the  needle  below  the  limits  of  the  bladder 
we  avoid  injuring  any  vital  part. 

Make  the  injections  at  intervals  of  five  days  and  give  increasing  amounts  at  each 
successive  injection.  Thus,  first  injection,  5  c.c.;  second  injection,  8  c.c.;  third 
injection,  10  c.c.;  fourth  injection  12  c.c.;  and  at  the  fifth  injection  give  about  15  to 
20  c.c.  of  washed  red-cell  sediment.  It  is  well  to  dilute  the  cell  sediment  with  an 
equal  amount  of  salt  solution.  About  ten  days  after  the  last  injection,  we  take  some 
blood  in  a  Wright's  tube  from  a  vein  of  the  ear  and  dilute  the  serum  to  make  a  i  to 
100  dilution.  To  i  c.c.  of  a  i%  emulsion  of  red  cells  we  add  o.i  c.c.  of  a  20%  dilu- 
tion of  guinea-pig's  fresh  serum — similar  combinations  being  made  in  a  series  of 
10  tubes.  To  each  of  these  tubes  we  add  varying  amounts  of  the  i  to  100  dilution, 
o.i  c.c.  in  the  first,  0.2  c.c.  in  the  second,  0.3  c.c.  in  the  third,  and  so  on.  If  we 
obtain  haemolysis  in  the  tube  containing  0.2  c.c.  of  i  to  100  dilution  of  serum  but  not 
in  that  containing  o.i  c.c.  we  note  that  the  serum  has  a  titre  of  about  i  to  500.  If 
the  o.i  c.c.  gave  haemolysis,  the  serum  would  have  a  titer  of  i  to  1000. 

Having  ascertained  that  the  haemolytic  serum  is  sufficiently  strong  we  shave  the 
left  side  of  the  thorax  of  the  rabbit  and  enter  the  needle  of  the  apparatus  similar  to 
that  used  for  taking  the  blood  from  a  man's  vein  in  one  of  the  intercostal  spaces  of  the 
left  side. 

Having  introduced  the  needle,  feel  for  the  heart  beat  and  then  plunge  the  needle 
into  the  heart.  We  can  withdraw  about  30  c.c.  of  blood  without  injury  to  the  rabbit. 
This  blood  should  be  received  in  a  clean  empty  flask  and  set,  over  night,  in  the  re- 
frigerator. The  following  morning  pour  off  the  clear  serum  into  a  clean  Petri  dish 
and  saturate,  one  by  one,  squares  of  filter  paper  with  the  serum.  Allow  the  filter 
paper  to  dry  on  a  piece  of  unbleached  muslin.  Noguchi  recommends  Schleich  and 
SchulPs  paper  No.  597.  When  thoroughly  dry  cut  strips  10  mm.  wide.  This  makes 
the  amboceptor  paper.  To  standardize,  take  a  series  of  tubes  containing  i  c.c.  of 
a  i%  emulsion  of  red  cells  and  add  o.i  c.c.  of  20%  dilution  of  guinea-pig  serum  for 
complement.  Next  cut  across  the  amboceptor  paper  strip  pieces  of  varying  width, 


WASSERMANN   TESTS  153 

as  i  mm.,  2  mm.,  3  mm.,  5  mm.,  and  so  on.  The  narrowest  strip  which  gives  hasmo- 
lysis  in  one  hour  equals  one  unit.  Thus  if  a  piece  5  mm.  wide  was  required  to  pro- 
duce haemolysis,  5  mm.  of  the  paper  would  have  a  value  of  one  unit. 


NOGUCHI'S  METHOD. 

For  the  suspension  of  red  cells  use  a  1/2%  suspension  of  washed 
human  red  cells. 

For  complement  use  fresh  guinea-pig  serum  in  a  dilution  of  i  part 
to  i  1/2  parts  of  salt  solution  (40%). 

Experiment. — 'Take  4  small  test-tubes  (12  by  125  mm.)  label  id,  ib 
and  20,  2b,  respectively.  Into  la  and  ib  each  put  i  drop  of  the  serum 
of  the  patient  to  be  tested  and  into  ia  and  2b  each  put  i  drop  of  the 
serum  of  a  person  known  to  give  a  positive  test  for  syphilis.  Next 
add  to  each  of  all  four  tubes  i  c.c.  of  the  1/2%  suspension  of  washed 
red  cells.  Then  add  to  each  tube  o.i  c.c.  of  the  40%  fresh  guinea-pig 
serum.  Now  add  to  tube  la  and  tube  ia  each  o.i  c.c.  of  the  i  to  10 
antigen  dilution  (opalescent  working  antigen  emulsion).  Tubes  ib  and 
2b  are  controls  not  containing  antigen.  Mix  contents  of  tubes  thor- 
oughly and  incubate  at  37°  C.  for  one  hour  or  for  1/2  hour  in  a  water 
bath.  Now  add  to  each  of  the  four  tubes  2  units  of  the  immune 
haemolytic  serum,  as  measured  off  on  the  amboceptor  paper  strip — thus 
with  a  paper  of  which  2  mm.  equals  i  unit,  drop  into  each  tube  4  mm. 
of  the  strip. 

The  tubes  without  antigen  (ib  and  26)  should  show  good  haemolysis. 
Tube  20,  that  of  the  known  syphilitic,  with  antigen,  should  not  show 
haemolysis  and  that  of  the  person  examined  (10)  should  show  haemolysis 
in  case  the  test  is  negative  for  syphilis.  Moderately  positive  cases  may 
show  a  slight  trace  of  haemolysis.  In  case  the  tubes  without  antigen  are 
negative  (no  haemolysis),  repeat  the  test  with  smaller  amounts  of  human 
serum.  It  may  be  advisable  to  employ  the  serum  of  a  person  known  to 
be  free  from  syphilis.  In  this  case  we  should  use  two  additional  tubes, 
30  and  3#,  conducting  the  test  as  for  the  syphilitic  control  serum. 

Many  workers  prefer  to  use  inactivated  serum  for  the  test.  In  this  case  we  should 
add  four  times  as  much  of  the  inactivated  serum  as  for  the  unheated  serum. 

Inactivation  not  only  destroys  complement  but  likewise  diminishes  the  strength 
of  the  antibody  content  of  the  serum.  Factors  such  as  character  of  food  and  general 
condition  influence  the  complement  strength  of  guinea-pig  serum  so  that  it  is  ad- 
visable to  titrate  the  guinea-pig  serum.  To  do  this  take  i  c.c.  of  a  i%  emulsion  of 
human  red  cells  and  drop  in  one  unit  of  amboceptor  paper.  The  amount  of  comple- 


154  PRACTICAL   METHODS   IN   IMMUNITY 

ment  which  will  entirely  haemolize  the  red  cells  in  one-half  an  hour  in  water  bath 
equals  one  unit  of  complement.     For  the  test  use  two  units  of  complement. 


THE  WASSERMANN  TEST. 

In  the  Wassermann  reaction  the  rabbits  are  injected  with  sheep 
red  cells  which  have  been  washed  twice  with  salt  solution  by  aid  of  the 
centrifuge.  About  five  injections  with,  on  the  average,  the  quantity  of 
red  cells  contained  in  5  c.c.  of  sheep  blood  given  at  intervals  of  five  days 
gives  a  strong  haemolytic  serum  if  taken  about  one  week  after  the  last 
injection.  The  method  is  to  take  in  a  test-tube  0.2  c.c.  inactivated 
human  serum  (heated  for  one  hour  at  56°  C.),  o.i  c.c.  fresh  serum  from 
guinea-pig  for  complement,  i  unit  antigen  and  3  c.c.  normal  salt  solu- 
tion; then  to  incubate  for  one  hour  at  37°  C.  (An  antigen  unit  is  the 
amount  that  will  inhibit  haemolysis  of  i  c.c.  of  5%  emulsion  of  sheep 
cells  when  mixed  with  0.2  c.c.  luetic  serum  and  o.i  c.c.  guinea-pig  comple- 
ment.) Then  add  2  units  of  amboceptor  and  i  c.c.  5%  emulsion  of 
sheep  red  cells,  shake  and  incubate  for  one  hour.  (The  amount  of 
haemolytic  serum  that  will  haemolize  i  c.c.  of  a  5%  emulsion  of  sheep 
red  cells  to  which  o.i  c.c.  guinea-pig  serum  has  been  added,  in  one  hour, 
is  an  amboceptor  unit.) 

The  same  technic  is  employed  with  the  control  test-tube  except  that 
the  antigen  unit  is  not  put  in. 

The  Noguchi  method  gives  a  positive  reaction  with  nonsyphilitic 
sera  in  about  7%  of  cases.  The  Wassermann  gives  a  negative  result 
in  about  9%  of  syphilitic  sera.  These  figures  show  the  advantage  of 
checking  one  against  the  other. 

There  seem  to  be  certain  sera  when  with  a  clinical  history  of  syphilis  we 
obtain  a  positive  Wassermann  with  unheated  serum  and  a  negative  one  with 
inactivated  serum.  In  order  to  obtain  information  with  the  same  serum  heated 
and  unheated  I  would  recommend,  when  it  is  inconvenient  to  carry  out  the  original 
Wassermann  technic,  to  employ  the  Noguchi  technic  with  inactivated  serum  and 
the  Emery  technic  with  fresh  unheated  serum.  In  any  case  when  serum  cannot  be 
tested  within  twenty-four  hours  it  should  be  inactivated,  as  unheated  serum  tends 
to  become  anticomplementary. 

Cherry  thinks  anticomplementary  bodies  are  found  during  chloroform  anaes- 
thesia. If  the  antigen  should  also  have  anticomplementary  action  the  total  might 
give  a  negative  result. 

By  heating  the  serum  for  half  an  hour  at  56°  C.  (inactivation)  the  positive 
results  obtained  in  certain  cases  of  cancer,  nephritis,  scarlet  fever,  leprosy  and  tuber- 
culosis may  be  avoided;  the  syphilitic  antibody  alone  being  thermostable.  The 


WASSERMANN   TESTS  155 

thermostability  of  serum  of  inherited  syphilis  is   the  highest — that  of  primary 
syphilis  the  least  of  luetic  sera. 

McDonagh  states  that  in  the  primary  stage  the  Wassermann  is 
positive  in  40%  of  cases.  In  secondary  cases  97%  give  positive  results 
when  treatment  has  not  been  instituted.  In  tertiary  syphilis  about 
70%  are  positive. 

In  268  cases  at  the  medical  clinic  of  Johns  Hopkins  Hospital,  Clough 
failed  to  obtain  a  positive  reaction  in  ninety-nine  cases  which  were 
negative  clinically. 

In  forty-five  cases  of  syphilis  he  obtained  73%  of  positive  results. 
Excluding  cases  which  had  received  thorough  treatment  82%  were 
positive.  Tabes  gave  40%  and  general  paresis  100  %.  In  five  cases 
of  primary  syphilis  four  gave  positive  reactions. 

Based  upon  the  observation  of  Bauer,  that  human  serum  contains  haemolytic 
amboceptors  for  sheep  corpuscles,  and  of  Hecht,  that  the  complement  normally 
present  in  human  serum  would  suffice  without  the  addition  of  guinea-pig  serum 
complement,  the  following  method  of  Fleming  is  easy  of  application. 

For  the  test  we  use: 

1.  Alcoholic  extract  of  rabbit's  heart,  made  by  washing  the  recently  removed 
heart  with  salt  solution  to  remove  all  blood.     Cut  into  small  pieces  and  grind  in  a 
mortar  with  sand  and  for  every  gram  of  heart  add  5  c.c.  of  95%  alcohol.     Keep  the 
mixture  at  a  temperature  of  60°  C.  for  two  hours  and  filter.     This  is  the  stock 
solution.     For  use  dilute  it  ten  times  with  normal  salt  solution. 

2.  A  5%  emulsion  of  washed  sheep  red  cells,  prepared  as  for  the  Wassermann 
test. 

3.  Suspected  and  control  sera. 

With  a  capillary  bulb  pipette  take  up  one  part  of  serum  and  four  parts  of  the 
heart  antigen,  mix  on  a  glass  slide,  again  draw  up  into  the  capillary  pipette  and, 
leaving  a  separating  air  space,  next  draw  up  one  part  of  5%  emulsion  of  sheep  red 
cells.  Then  seal  off  tip  of  pipette  and  incubate  at  37°  C.  for  one  hour.  Now  file 
off  tip  and  mix  the  red  cells  with  the  serum  and  antigen  and  again  draw  up  into  the 
capillary  pipette  and  incubate  a  second  time  for  two  hours.  Haemolysis  or  the 
reverse  is  shown  in  the  fluid  overlying  the  cell  sediment.  Various  controls  should 
be  made  using  normal  and  known  syphilitic  sera;  also  with  normal  salt  instead  of 
serum. 

The  objections  to  methods  using  human  serum  for  complement  are 
i.  the  great  variation  in  the  complement  content  of  different  human 
sera;  2.  human  complement  requires  about  ten  times  as  much  ambo- 
ceptor  as  guinea-pig  complement  and  is  less  sensitive  to  fixation,  and 
3,  the  statement  is  made  by  s>me  workers  that  while  homologous 
complement  and  amboceptor  may  be  efficient  yet  the  complement  of  a 


156  PRACTICAL   METHODS    IN   IMMUNITY 

serum  will  not  act  upon  its  homologous  antigen.  This  is  not  true 
because  the  complement  of  human  serum  invariably  haemolyzes  the 
homologous  antigen  (human  red  cells). 

The  various  precipitate  tests  that  have  been  proposed  are  unreliable.  The 
precipitate  reactions  with  bile  salts  give  better  results  than  with  lecithin,  this  latter 
showing  positive  results  in  almost  one-half  of  non-syphilitic  cases. 

DETERMINATION  or  OPSONIC  POWER  AND  THE  PREPARATION 
or  VACCINES. 

In  that  which  has  been  considered  in  the  previous  pages  only  the 
theories  of  Ehrlich  have  been  brought  out.  In  order  to  understand  the 
problems  involved  in  the  study  of  opsonins  the  phagocytic  theory  of 
immunity  brought  forward  by  Metchnikoff  must  be  studied.  Ehrlich's 
views  would  seem  to  hold  with  diseases  where  there  is  an  increase  in 
bacteriolytic  or  antitoxic  power  of  the  serum  while  in  such  diseases,  as 
are  caused  by  pathogenic  cocci,  the  phagocytic  element  is  operative  as 
there  is  an  absence  of  bacteriolytic  power  in  the  serum  of  the  person 
with  the  infection. 

There  are  two  kinds  of  phagocytes,  the  microphages  (represented 
by  the  polymorphonuclears)  which  on  phagolysis  or  disintegration  give 
off  microcytase,  a  bactericidal  substance.  Cytase  is  the  same  as 
complement  or  alexine. 

The  microphages  are  chiefly  bactericidal  while  the  macrophages, 
represented  by  the  large  mononuclears  of  the  blood  and  fixed  connective- 
tissue  cells,  exert  their  action  on  protozoa  or  animal  cells. 

Phagocytes  may  either  act  by  ingesting  bacteria  and  destroying 
them  intracellulary  or  they  may  as  a  result  of  phagolysis  bring  about 
bacteriolysis  extracellularly.  According  to  Metchnikoff  the  intra- 
cellular  bacteriolysis  explains  why  an  individual  may  possess  immunity 
and  yet  his  serum  fail  to  show  any  bacteriolytic  power. 

The  following  modification  of  Leishman's  method  takes  very  little  time  and  skill 
and  is  applicable  in  the  determination  of  the  organism  concerned  in  an  infection,  as 
in  Wright's  method.  The  control  of  vaccine  treatment  by  taking  opsonic  indices 
from  time  to  time  does  not  seem  to  have  met  with  much  favor  in  this  country — the 
sources  of  error  being  as  great,  if  not  greater,  than  ordinary  variations  in  the  opsonic 
index  during  the  negative  and  positive  phases. 

Method. — We  start  with  a  i%  solution  of  sodium  citrate  in  salt  solution.  With 
this  emulsify  a  twelve  to  twenty-four-hour  agar  slant  growth  of  the  organism  to  be 
tested  using  6  to  8  c.c.  of  the  citrated  salt  solution.  The  bacterial  emulsion  is  now 


OPSONIC   INDEX  157 

poured  into  a  bottle  or  sealed  off  in  a  test-tube  and  shaken  thoroughly  in  a  shaker  or 
by  hand.  The  emulsion  is  then  centrifuged  to  throw  down  the  bacterial  clumps  and 
the  supernatant  slightly  turbid  bacterial  suspension  poured  off.  If  working  with  a 
dangerous  pathogen  it  is  advisable  to  kill  the  organisms  as  in  making  vaccines. 

Now  with  a  capillary  bulb  pipette  so  graduated  that  the  one  volume  mark  con- 
tains about  two  drops  we  draw  up  one  volume  of  citrated  salt  solution.  Then 
having  made  a  break  with  an  air  column,  we  take  up  one  volume  of  the  patient's 
blood.  Again  make  an  air  break  and  draw  up  one  volume  of  the  citrated  salt  solu- 
tion bacterial  emulsion.  The  three  volumes  are  then  immediately  forced  out  into 
a  small  test-tube,  made  from  three  inches  of  3/16- inch  glass  tubing,  as  shown  in  the 
Emery  technic  for  the  Wassermann.  The  citrate  prevents  coagulation  of  the  blood 
and  the  contents  of  the  tube  are  well  mixed  by  drawing  up  and  ejecting  with  the 
capillary  bulb  pipette.  Incubate  this  small  test-tube  at  body  temperature  for 
15  minutes,  shaking  the  contents  once  or  twice  during  the  incubation  period.  Ex- 
actly at  the  expiration  of  the  period  of  incubation  (usually  15  minutes  although  at 
times  10  minutes  or  30  minutes  may  be  desirable)  place  the  tube  in  a  centrifuge  and 
throw  down  the  cell  sediment.  Next  pipette  off  the  supernatant  fluid  and  then 
plunge  the  pipette  to  the  bottom  of  the  tube  and  draw  off  the  greater  part  of  the 
sediment  at  the  bottom.  This  consists  largely  of  the  red  cells  the  leukocyte  layer 
on  the  surface  being  undisturbed. 

Now  mix  the  remaining  cell  sediment  and  smear  out  on  a  slide  or  preferably 
between  two  cover-glasses  as  in  Ehrlich's  method.  The  smear  is  fixed  by  burning 
off  a  film  of  alcohol  and  stained  with  dilute  carbol  fuchsin  or  methylene  blue.  The 
granule  staining  with  Wright's  stain  makes  it  slightly  confusing. 

A  second  similar  preparation  but  using  blood  from  a  normal  person  as  a  control 
is  then  made.  Counting  the  phagocytized  bacteria  in  a  given  number  of  poly- 
morphonuclears,  we  obtain  an  average  number  of  bacteria  phagocytized  per  cell. 
Repeating  the  count  with  the  control  or  normal  blood,  we  likewise  have  the  average 
number  of  bacteria  taken  up  per  cell.  Dividing  the  patient's  average  by  the  normal 
average,  we  have  the  opsonic  index.  If  the  average  for  fifty  of  the  patient's  cells  was 
eight  and  that  of  the  control  only  four,  the  patient's  index  would  be  two,  or  twice  the 
normal.  The  practical  value  of  this  test  is  that  where  two  or  more  organisms  are  in  a 
body  fluid  we  may  ascertain  the  causative  organism  by  noting  marked  variation 
from  the  normal  in  the  patient's  opsonic  index  for  that  particular  organism  and  not 
for  the  other  organism.  This  variation  may  be  of  the  nature  of  a  high  or  low  opsonic 
index. 


METHOD  or  WRIGHT  FOR  OBTAINING  OPSONIC  INDEX. 

While  other  observers  had  previously  noted  the  presence  of  sub- 
stances in  immune  sera  which  so  acted  on  the  bacteria  that  phagocytosis 
was  made  possible,  yet  it  was  to  Wright  and  Douglas,  in  1903,  that  the 
existence  of  this  factor  in  phagocytosis  was  brought  forward  and  the 
estimation  of  such  substances  made  practicable. 

To  this  substance  the  name  opsonin  was  given — 'the  Greek  word 


158  PRACTICAL   METHODS    IN    IMMUNITY 

from  which  it  is  derived  indicating  preparation  of  the  food — that  is, 
the  opsonin  so  alters  or  sensitizes  the  bacteria  that  they  can  be  engulfed 
or  phagocytized  by  the  polymorphonuclear  leukocytes  (the  microphages 
of  Metchnikoff).  About  the  same  time  Neufeld  and  Rimpau  noted 
the  presence  of  a  substance  in  immune  sera  which  so  acted  on  bacteria 
as  to  prepare  them  for  phagocytosis.  Their  designation"  bacterio- 
tropic  substance"  is  practically  synonymous  with  opsonin. 

In  1902  Leishman  introduced  the  method  of  determining  the  "phagocytic  index." 
By  taking  one  part  of  blood  and  one  part  of  an  emulsion  of  the  bacteria  in  question 
and  keeping  the  mixture  in  a  moist  chamber  at  body  temperature  for  a  standard 
time,  as  15  to  30  minutes,  and  then  spreading  the  blood-bacteria  mixture  and 
staining  the  film  with  Leishman  or  Wright's  stain  he  counted  the  number  of  bacteria 
in  a  certain  number  of  polymorphonuclears,  and  by  dividing  obtained  the  average 
number  per  leukocyte  of  bacteria  phagocytized. 

The  Wright  technic  for  determining  the  phagocytic  average,  and 
from  this  the  opsonic  index,  is  as  follows: 

Blood  is  taken  from  the  patient  and  at  the  same  time  from  a  normal  individual, 
or  preferably  the  blood  of  several  normal  individuals  is  pooled.  This  blood  is  best 
collected  in  a  Wright's  tube,  although  it  may  be  received  in  a  small  test-tube.  After 
coagulation  and  separation  of  the  serum,  the  serum  is  ready  for  use. 

The  next  step  is  to  prepare  the  leukocyte  emulsion.  For  this  we  fill  a  centri- 
fuge tube  with  normal  salt  solution,  to  which  has  been  added  i%  sodium  citrate 
— the  latter  to  prevent  coagulation.  Then  having  pricked  a  finger  congested  by  a 
constricting  rubber  band,  from  15  to  20  drops  of  blood  are  added  to  the  citrated 
salt  solution,  and  the  mixture  thoroughly  shaken.  After  centrifugalization  for  about 
5  minutes  the  red  corpuscles  will  be  thrown  to  the  bottom  of  the  tube  with  the  leu- 
kocytes forming  a  superimposed  layer.  In  order  to  free  the  leukocytes  entirely  from 
serum  admixture,  the  supernatant  citrated  salt  solution  is  pipetted  off,  and  a  fresh 
tubeful  of  salt  solution  is  added  to  the  blood-cell  sediment.  Again  shaking,  we  then 
centrifuge,  obtaining  for  a  second  time  a  sediment  of  blood  cells  with  the  leukocytes 
in  the  superimposed  layer.  In  some  laboratories  the  washing  in  salt  solution  is 
again  repeated,  but  for  all  practical  purposes  two  washings  as  described  above  suffice. 

The  superimposed  layer  of  white  cells  may  now  be  pipetted  off  from  the  heavier 
red  cells  (of  course,  containing  a  large  admixture  of  red  cells)  to  be  used  as  a  leuko- 
cyte cream — or  by  slanting  the  centrifuge  tube  we  can  pipette  off  the  proportion 
of  the  leukocyte  mixture  needed  from  the  bottom,  sides  or  top  of  the  slanted  layer 
of  blood  cells. 

Having  prepared  our  leukocyte  emulsion,  and  the  serum  from  the  normal  in- 
dividual as  well  as  that  from  the  patient,  it  only  remains  to  prepare  our  bacterial 
emulsion.  For  bacteria  in  general,  with  the  exception  of  tubercle  bacilli,  we  simply 
take  up  a  small  loopful  of  a  young  agar  culture  (eighteen  hours  or  less),  and  emulsify 
it  uniformly  with  salt  solution,  added  by  degrees  until  the  suspension  amounts  to 
1/2  to  i  c.c.,  and  giving  a  faint  turbidity.  To  thoroughly  distribute  and  especially 


BACTERIAL  VACCINES  159 

to  break  up  clumps  repeated  suction  and  ejection  with  a  capillary  pipette  provided 
with  a  rubber  nipple  is  satisfactory. 

The  presence  of  clumps  in  a  bacterial  emulsion  invalidates  the  estimation  of 
phagocytosis,  for  the  reason  that  a  leukocyte  will  take  up  a  clump  of  twenty  or  more 
bacilli  as  readily  as  one  separate  organism. 

Having  at  hand  (i)  the  suspension  of  leukocytes,  (2)  the  bacterial  emulsion, 
and  (3)  the  sera  of  the  patient  and  the  normal  individual,  we  are  ready  to  proceed 
with  the  test. 

Using  a  capillary  bulb  pipette  with  a  pencil  mark  to  indicate  i  volume  we  draw 
up  to  the  mark  (i)  the  leukocyte  cream.  Then  wiping  off  the  tip  of  the  pipette  we 
draw  up  this  volume  of  leukocyte  emulsion  about  one-half  an  inch  to  make  an  air 
break  between  this  and  (2)  i  volume  of  the  bacillary  emulsion.  Again  making  an 
air  space  we  draw  up  (3)  the  serum  of  the  normal  individual.  This  gives  3  columns  in 
the  capillary  tube  with  intervening  breaks  of  air.  We  next  eject  the  three  con- 
stituents into  a  watch-glass  and  thoroughly  mix  them  by  alternate  suction  and  ejec- 
tion with  the  tube  and  nipple.  When  mixed  we  draw  the  mixture  up  into  the  same 
capillary  tube,  seal  off  the  capillary  end  in  the  flame  and  put  in  an  incubator  for 
exactly  15  minutes. 

We  next  repeat  the  process  identically  except  that  the  patient's  serum  is  used  in- 
stead of  that  of  the  normal  individual. 

These  tubes  having  been  kept  at  the  same  temperature  for  the  same  length  of 
time  are  then  taken  out,  the  contents  blown  into  a  watch  glass,  mixed  thoroughly 
a  second  time,  and  then  a  smear  is  made — a  drop  of  the  mixture  being  deposited 
on  a  very  clean  slide  and  the  smear  made  by  a  second  narrower  slide  (by  cutting  off 
the  corner  of  the  slide)  which  is  drawn  along  in  a  zigzag  way.  The  smears  are  then 
stained  (Leishman's  or  Wright's  blood  stain  or  Ziehl-Neelson's  for  tubercle  bacilli) 
and  the  number  of  the  bacteria  in  from  fifty  to  one  hundred  leukocytes  counted. 
This  number  divided  by  the  number  of  cells  gives  the  phagocytic  average. 

The  phagocytic  average  of  the  patient's  tube  divided  by  that  of  the  normal  in- 
dividual's tube  gives  the  opsonic  index.  Thus,,  in  counting  100  cells  we  find  500 
phagocytized  cocci  in  the  patient's  tube,  giving  an  average  of  5,  and  in  the  normal 
individuals  blood  we  get  1000,  an  average  of  10.  Then  the  opsonic  index  would 
be  5 -Mo,  or  0.5. 

PREPARATION  OF  VACCINES. 

It  has  been  found  satisfactory  to  make  use  of  stock  vaccines  in 
gonorrhceal  and  tuberculous  affections.  In  treatment  of  tuberculosis 
Wright  prefers  Koch's  T.  R.  or  Neu  Tuberculin  in  doses  of  from  1/5000 
to  1/800  of  a  milligram.  Some  prefer  Koch's  more  recent  bazillen 
emulsion.  In  case  of  other  infections,  however,  and  preferably  with 
gonorrhceal  infections,  the  causative  organism  should  be  isolated  from 
pus,  sputum,  urine,  blood,  or  other  material  (autogenous  vaccine). 

In  the  making  of  vaccines  all  media  and 'apparatus  should  be  sterilized  with 
scrupulous  care  to  avoid  the  danger  of  tetanus  infection.  Having  isolated  the  organ- 


160  PRACTICAL'  METHODS   IN   IMMUNITY 

ism,  it  is  inoculated  upon  one  or  more  agar  slants,  and  after  a  growth  of  from  five 
to  seven  hours  with  streptococci  and  pneumococti,  or  with  eighteen  hours  for  staphy- 
lococci  and  colon,  the  growth  on  these  inoculated  slants  is  taken  up  with  salt  solu- 
tion, thoroughly  shaken  up  in  the  diluting  solution  and  standardized. 

The  most  practical  way  is  to  gently  rub  off  the  growth  on  the  agar  in  about  i  or 
2  c.c.  of  salt  solution  with  a  platinum  loop.  Then  pour  the  bacterial  emulsion  into 
a  sterile  test-tube  and  repeat  the  process  with  three  to  five  agar  slants,  until  we  have 
from  six  to  10  c.c.  of  the  emulsion  in  the  sterile  test-tube.  By  heating  to  melting- 
point  in  the  flame  a  piece  of  glass  tubing  and  attaching  it  to  the  rim  of  the  test- 
tube  (also  melted),  we  have  a  handle  with  which  to  draw  out  the  test-tube  when 
heated  about  i  inch  from  the  mouth  in  a  blowpipe  flame.  Drawing  this  out,  we  let  it 
cool,  and  then  filing  the  constricted  portion  we  break  it  off  and  seal  it  in  the  flame. 
By  shaking  up  and  down  vigorously  for  five  to  fifteen  minutes,  or  preferably  in  a 
mechanical  shaker  the  bacteria  are  distributed  evenly  in  the  salt  solution.  A  piece 
of  platinum  wire,  twisted  into  corkscrew  shape,  and  fused  in  the  drawn  out  end  of 
the  containing  test-tube  helps  in  breaking  up  the  bacterial  emulsion  and  is  a  great 
aid  in  the  preparation  of  streptococcic  or  diphtheroid  vaccines. 

The  sealed  test-tube  is  then  placed  in  a  water-bath  at  60°  C.  and  heated  at  this 
temperature  for  one  hour.  Again  shake.  The  constricted  sealed  end  is  again  filed 
off  and  a  few  drops  shaken  out  in  a  watch  glass  for  standardization,  and  at  the  same 
time  a  few  drops  are  deposited  on  an  agar  slant  as  a  test  for  sterility.  (Incubation 
for  twenty-four  hours  should  not  show  growth.) 

Wright  found  that  by  taking  a  definite  quantity  of  blood  and  a 
similar  quantity  of  bacterial  emulsion,  mixing  the  blood  and  bacterial 
emulsion,  then  making  a  smear  and  staining,  it  was  possible  to  de- 
termine the  ratio  of  bacteria  to  red  cells,  and  from  this  the  number  of 
bacteria  per  cubic  centimeter  could  be  determined.  For  example,  if 
we  find  three  bacteria  to  each  red  cell  we  should  have  15,000,000 
bacteria  to  i  cubic  millimeter.  (There  being  5,000,000  red  cells  to  the 
cubic  millimeter.)  As  i  cubic  centimeter  is  1000  times  greater  than  i 
cubic  millimeter,  there  would  be  15,000,000,000  bacteria  in  each  cubic 
centimeter  of  such  an  emulsion,  or  vaccine,  as  it  is  termed. 

The  standardization  made  with  a  haemacytometer  is  best  done  by  drawing  up 
the  vaccine  to  0.5  with  either  the  red  or  white  pipette,  according  to  concentration, 
and  then  sucking  up  i  to  10  dilute  carbol  fuchsin  to  n  or  101.  Allow  the  bacteria 
to  settle  on  the  shelf  for  ten  minutes  before  counting.  Count  as  in  making  a  red 
count. 

A  more  satisfactory  diluting  fluid  is  that  recommended  by  Callison.  It  is: 
Hydrochloric  acid  2  c.c.,  Bichloride  of  mercury  (1-500  aq.  sol.)  100  c.c.,  and  sufficient 
i%  aqueous  solution  of  acid  fuchsin  to  color  the  diluting  mixture  a  deep  cherry  red. 
The  diluting  fluid  should  then  'be  filtered.  The  bichloride  forms  an  albuminate  on 
the  surface  of  the  bacteria  which  promotes  rapid  sedimentation  and  the  fuchsin 
stains  the  bacteria. 


BACTERIAL  VACCINES  l6l 

Having  determined  the  strength  of  the  stock  vaccine,  we  should 
prepare  a  dilute  vaccine  for  injection.  This  is  most  conveniently 
carried  out  by  filling  vials  with  50  c.c.  of  salt  solution,  plugging  with 
cotton,  then  sterilizing  in  the  autoclave.  A  sterile  rubber  cap  is  now 
drawn  over  the  mouth  of  the  vial.  Sterility  is  insured  by  plunging  the 
rubber  cap  and  neck  in  boiling  water.  If  the  stock  vaccine  showed 
5,000,000,000  bacteria  per  c.c.  and  we  desired  to  have  a  vaccine  con- 
taining 200,000,000  bacteria  per  c.c.,  it  would  be  necessary  to  draw  out 
2  c.c.  of  the  salt  solution  by  means  of  a  sterile  .syringe  needle  inserted 
through  the  rubber  cap  and  replace  it  with  2  c.c.  of  the  bacterial 
emulsion.  Example:  In  introducing  2  c.c.  of  a  vaccine  containing 
5,000,000,000  bacteria  per  c.c.,  we  throw  in  10,000,000,000  bacteiia  in 
a  volume  equal  to  50  c.c.  Then  each  c.c.  of  the  50  c.c.  in  the  bottle 
would  contain  10,000,000,000  divided  by  50  or  200,000,000  in  each  c.c. 
If  we  only  want  a  vaccine  containing  100,000,000  per  c.c.  we  should  only 
add  i  c.c.  We  now  add  1/4%  of  trikersol  to  the  vaccine  in  order  to 
insure  sterility.  (Introduced  with  syringe,  inserting  needle  through 
rubber  cap.)  The  syringe  is  best  sterilized  by  drawing  up  vaseline 
or  olive  oil  heated  to  150°  C.,  and  the  neck  and  rubber  cap  of  the 
bottle  in  boiling  water.  We  now  draw  up  the  desired  dose  of  bacteria. 
If  glass  syringes  are  used,  simply  boiling  in  water  suffices.  The 
ordinary  doses  are:  For  gonococci,  streptococci,  pheumococci,  and 
colon  vaccines,  5,000,000  to  50,000,000.  For  staphylococci  200,000,000 
to  i, 000,000,000. 

Wilson  gives  the  following  minimum  and  maximum  doses  expressed  in  millions: 

Streptococcus,  6  and  68. 

Gonococcus,  45  and  900. 

Meningococcus,  300  and  900. 

M.  melitensis,  700  and  1400. 

B.  coli,  16  and  240. 

B.  typhoid  (treatment)  100  and  250. 

B.  typhoid  (prophylaxis)  500  and  1000. 

B.  pyocyaneus,  34  and  1000. 

B.  pneumoniae,  44. 

Staphylococci,  150  and  900. 

B.  tuberculosis,  1/20000  to  1/200  milligram. 

ANAPHYLAXIS. 

This  is  a  term  which  indicates  the  opposite  of  prophylaxis.     It  was 
noted  that  after  a  period  of  incubation  of  at  least  ten  days  a  second 


1 62  PRACTICAL    METHODS    IN    IMMUNITY 

injection  of  horse  serum  produced  symptoms  of  respiratory  embarrass- 
ment, convulsions  and,  at  times,  death.  The  primary  injection  had 
during  the  period  of  incubation  sensitized  the  cells  to  this  particular 
proteid. 

This  phenomenon  of  sensitization  in  the  case  of  rabbits  bears  the 
name  of  Arthus,  and  as  applied  to  guinea-pigs  sensitized  with  diphthe- 
ria antitoxin  sera  the  name  Theobald  Smith,  and  it  is  stated  by  Muir 
and  Ritchie  that  active  research  as  to  anaphylaxis  may  be  said  to  date 
from  the  discovery  of  the  phenomenon  of  Theobald  Smith. 

Rosenau  and  Anderson  working  with  guinea-pigs  showed  that  small 
doses  were  efficient  for  sensitization,  that  the  condition  was  trans- 
missible from  mother  to  offspring  and  that  a  second  animal  could  be 
sensitized  by  being  injected  with  the  serum  of  a  sensitized  animal. 

This  group  of  symptoms,  the  so-called  anaphylactic  shock,  which  is  apt  to  set 
in  within  a  few  minutes  after  the  second  injection,  is  often  preceded  by  restlessness 
and  great  excitement  and  together  with  the  dyspnoeic  manifestations  there  is  cardiac 
weakness  and  great  fall  of  blood-pressure.  The  more  serious  symptoms  and  at  times 
death  are  more  apt  to  appear  after  intracerebral  injections  than  after  intraperitoneal. 
Subcutaneous  injections  are  least  apt  to  produce  anaphylactic  symptoms.  Our 
attention  to  this  phenomenon  commenced  with  the  study  of  "serum  sickness"  or 
"serum  disease."  In  this  an  erythematous  rash  or  urticaria  associated  with  more 
or  less  oedema  comes  on  after  eight  to  twelve  days  from  the  time  of  the  first  and  only 
injection  of  horse  serum.  It  is  supposed  to  be  due  to  the  fact  that  some  of  the  serum 
originally  injected  remains  unchanged  in  the  tissues  so  that  when  the  sensitization 
takes  place  there  is  present  and  at  hand  the  same  foreign  proteid  to  bring  about 
anaphylactic  symptoms. 

Immunization  against  anaphylaxis  is  possible  by  repeating  injection  of  the  sen- 
sitizing serum  or  proteid  during  the  period  of  incubation. 

It  is  important  to  note  that  this  hypersusceptibility  appears  to  be 
very  rarely  of  importance  in  the  matter  of  the  administration  of  a 
second  injection  of  diphtheria  antitoxin  after  the  period  of  anaphylactic 
incubation. 

As  a  rule  the  death  or  untoward  effects  of  the  injection  of  serum  are 
in  cases  of  status  lymphaticus.  Cases  in  man  do  occur,  however,  but 
with  extreme  infrequency,  in  which  within  a  few  minutes  after  the  only 
injection  of  serum  the  patient  becomes  restless,  shows  symptoms  of 
cardiac  and  repiratory  embarrassment  and  may  be  dead  in  a  very  short 
time. 

According  to  Rosenau  and  Anderson  individuals  who  have  asthmatic  tendencies 
as  well  as  those  who  have  had  serum  injections  ten  to  twelve  days  or  longer  prior  to 


ANAPHYLAXIS  163 

the  second  injection  should  be  considered  as  possible  subjects  for  anaphylactic 
shock. 

Vaughan  recommends  that  when  this  is  to  be  feared  one  should  only  give  about 
o.i  c.c.  of  the  serum  and  after  an  interval  of  two  hours,  provided  no  untoward  symp- 
toms set  in,  to  give  the  full  amount  of  the  injection.  Besredka  advises  heating  the 
serum  to  56°  as  a  guard  against  anaphylactic  shock. 

The  condition  of  hypersusceptibility  or  anaphylaxis  is  at  times 
termed  allergy.  Thus  in  a  person  who  has  been  successfully  vacci- 
nated a  reaction  shows  at  the  site  of  inoculation  within  twenty-four 
hours  which  does  not  appear  in  the  nonimmune  person  for  a  period  two 
or  three  times  as  long.  The  diagnostic  tests  with  tuberculin  and 
luetin  are  hence  often  referred  to  as  allergic  reactions. 

It  may  here  be  stated  that  some  investigators  are  of  the  opinion  that 
our  views  not  only  as  to  immunity  but  as  to  the  essential  nature  of 
infectious  diseases  may  be  later  on  found  to  rest  in  production  of 
anaphylaxis. 

The  name  "Anaphylactine"  has  been  applied  to  the  sensitizing  substance  pro- 
duced during  the  period  of  incubation. 

It  has  been  proposed  to  employ  this  phenomenon  as  a  diagnostic  measure.  By 
taking  the  serum  of  a  tuberculous  patient,  which  would  contain  the  sensitizing  sub- 
stance, and  injecting  it  into  the  peritoneal  cavity  of  a  rabbit,  the  animal  would 
be  sensitized  and  an  injection  of  tuberculin  a  few  hours  later  would  bring  about  the 
phenomena  of  anaphylaxis  in  the  rabbit. 

This  passive  anaphylaxis,  as  it  is  termed,  usually  requires  approximately  twenty- 
four  hours  for  sensitization.  This  passive  anaphylactic  sensitization  seems  to  dis- 
appear in  two  weeks.  It  has  been  advised  to  passively  sensitize  guinea-pigs  with 
the  serum  of  the  person  about  to  be  injected  and  then  twenty-four  hours  after  inject 
the  guinea-pigs  with  the  curative  serum.  If  untoward  results  occur  in  the  guinea- 
pigs  the  patient  should  not  receive  the  injection. 

Recently  Hagemann  has  found  the  following  technic  valuable  in  the  diagnosis 
of  surgical  tuberculosis.  Guinea-pigs  are  inoculated  intraperitoneally  with  tuber- 
culosis cultures  and  by  the  end  of  the  second  week  such  pigs  are  sensitized.  The 
suspected  material,  as  serous  effusion,  is  injected  intracutaneously  and  within 
twenty-four  to  forty-eight  hours  a  distinct  swelling  of  the  skin  with  a  bluish-red 
center,  which  is  surrounded  by  a  porcelain  white  ring  and  outside  of  this  a  zone 
of  inflammation,  shows  a  positive  test. 


NOTES  ON  BACTERIOLOGY. 


NOTES  ON  BACTERIOLOGY. 


NOTES  ON  BACTERIOLOGY. 


NOTES  ON  BACTERIOLOGY. 


NOTES  ON  BACTERIOLOGY. 


PART  II. 
STUDY  OF  THE  BLOOD. 

CHAPTER  XIII. 
MICROMETRY  AND  BLOOD  PREPARATIONS. 

MlCROMETRY. 

IN  the  examination  of  blood  and  faeces  preparations,  especially 
when  the  identification  of  animal  parasites  is  in  question,  there  is  noth- 
ing that  assists  more  than  a  knowledge  of  the  measurements  of  the  object 
studied.  The  making  of  such  measurements  microscopically  is  termed 
micrometry. 

Micrometry  is  also  indispensable  in  bacteriology  and  cytodiagnosis 
as  well  as  in  animal  parasitology. 

The  most  practical  way  of  making  these  measurements  is  with  an  ocular  microme- 
ter. These  can  be  bought  separately,  or  a  glass  disc  (disc  micrometer)  with  lines 
ruled  on  it  can  be  dropped  into  the  ocular  to  rest  on  the  diaphragm  inside  the  ocular. 
The  ruled  surface  of  this  glass  diaphragm  should  be  placed  downward.  As  was 
stated  in  connection  with  the  microscope,  the  image  of  the  object  is  formed  at  the 
level  of  the  diaphragm  rim  inside  the  ocular,  consequently  the  lines  of  the  image  cut 
those  of  the  lines  ruled  on  the  glass  in  the  ocular.  Once  having  standardized  the 
value  of  the  spaces  of  the  ocular  micrometer  for  each  different  objective,  all  that  is 
necessary  subsequently  in  measuring  is  to  count  the  number  of  lines  or  spaces  which 
the  image  of  the  object  fills  and  then,  knowing  the  value  of  each  space  for  that  ob- 
jective, to  multiply  the  number  of  spaces  by  the  value  of  a  single  space. 

The  unit  in  micrometry  is  the  micron.  This  is  usually  written  p 
and  is  the  i/iooo  part  of  a  millimeter.  There  are  1000  microns  in  a 
millimeter. 

To  standardize:  For  this  purpose  it  is  necessary  to  have  a  scale 
of  known  measurements.  The  stage  micrometers  are  usually  ruled 
in  spaces  of  o.i  and  o.oi  mm.  The  lines  which  are  i/io  of  a  milli- 
meter apart  are  consequently  separated  by  a  distance  of  100  microns; 
those  i/ 100  of  a  millimeter  apart  are  separated  by  a  distance  of  10 
microns. 

169 


iyo 


MICROMETRY   AND   BLOOD    PREPARATIONS 


The  ocular  micrometer  is  usually  ruled  with  50  or  100  lines  or  spaces, 
separated  by  longer  lines  into  groups  of  5  and  10. 

Having  brought  the  lines  on  the  stage  micrometer  to  a  focus,  we 
determine  the  number  of  spaces  on  the  stage  micrometer  which  the  50 
divisions  of  the  ocular  micrometer  cover.  To  distinguish  the  ruling  of 
the  ocular  from  that  of  the  stage  micrometer,  revolve  the  ocular  with 
the  fingers. 


FIG.  50. — Micrometry  diagrams,  i.  Ocular  micrometer  with  stage  micrometer. 
50  spaces  of  ocular  micrometer  cover  two  100  micron  spaces  and  ten  10  micron  spaces; 
equal  300  microns.  Each  division  on  ocular  micrometer  equals  6  microns.  2. 
Ocular  micrometer  subtending  image  of  whip  worm  egg.  9  spaces  of  ocular  mi- 
crometer cover  Whipworm  egg.  Each  space  equals  6  microns.  Whipworm  egg 
equals  54  microns.  3.  Ocular  micrometer  with  ruling  of  haemacytometer.  50 
spaces  of  ocular  micrometer  cover  space  equal  to  width  of  6  small  squares  50X6  =  300 
microns.  Each  division  of  ocular  micrometer  equals  6  microns. 

The  tube  length  which  is  used  at  the  time  of  standardizing  must 
always  be  adhered  to  in  subsequent  measurements. 

Example :  With  a  2/3-inch  objective,  the  50  rulings  of  the  ocular  micrometer 
fill  in  fifteen  of  the  i/io  millimeter  rulings  (ioo/<)  and  three  of  the  i/ioo  millimeter 
spaces  (IQ/X).  Consequently  the  50  spaces  of  the  ocular  cover  1530  microns  (15  X 100 
=  1500;  3X10  =  30).  Then  if  50  spaces  equal  1530  microns,  one  space  would  equal 


BLOOD   PREPARATIONS  171 

30.6  microns.  With  the  i/6-inch  objective  the  50  ocular  spaces  would  cover  about 
three  of  the  i/io  millimeter  (ioo/*)  spaces  of  the  stage  micrometer.  Then  the  50 
spaces  would  equal  300  microns  and  one  space  would  equal  6  microns. 

The  ruling  of  the  slide  of  a  Thoma-Zeiss  haemocytometer  will  answer  as  well  as 
a  stage  micrometer.  The  small  squares  are  1/20  of  a  millimeter  square,  consequently 
the  distance  between  the  lines  bordering  the  small  square  is  1/20  millimeter  or  50 
microns. 

Now,  if  with  the  i /6-inch  objective,  the  50  lines  on  the  ocular  fill  in  the  spaces  of 
six  small  squares,  the  length  of  such  a  space  would  be  50X6  =  300  microns.  This 
divided  by  50  spaces  would  equal  6/*. 

Should  there  be  100  spaces  on  the  ocular  micrometer  instead  of  50,  it  would 
only  be  necessary  to  divide  the  length  in  microns  of  the  ruled  surface  of  the  stage 
micrometer  covered  by  the  100  lines  of  .the  ocular  micrometer  by  100.  The  quotient 
would  give  the  value  in  microns  of  each  space  of  such  an  ocular  micrometer. 

The  most  accurate  instrument  for  measuring  is  the  filar  micrometer.  These  are 
expensive.  Measurements  can  also  be  made  with  the  camera  lucida,  but  it  takes 
considerable  time  to  make  the  adjustments  necessary,  so  that  it  is  not  convenient. 
With  an  ocular  micrometer  one  can  make  measurements  of  blood-cells,  amoebae,  etc., 
in  a  few  seconds — it  only  being  necessary  to  slip  in  the  ocular  micrometer. 

Rule  for  determining  the  magnifying  power  of  microscopic  lenses:  Measure  the 
diameter  of  the  lens  of  the  objective  in  inches — the  approximate  equivalent  focal 
distance  is  about  twice  the  diameter.  Dividing  10  by  the  equivalent  focal  distance 
gives  the  magnifying  power  of  the  lens.  This  should  be  multiplied  by  the  number 
of  times  the  ocular  magnifies.  Example:  The  diameter  of  the  lens  of  the  objective 
was  found  to  measure  1/2  inch,  the  focal  distance  would  then  be  about  i  inch. 
Dividing  10  by  i  we  have  10  as  the  magnifying  power  of  the  lens  of  the  objective. 
If  we  were  using  a  No.  4  ocular,  the  magnifying  power  would  be  approximately  forty. 


BLOOD  PREPARATIONS. 

To  obtain  blood,  except  for  blood  cultures,  use  either  a  platino- 
iridium  hypodermic  needle  which  can  be  sterilized  in  the  flame,  a  small 
lancet,  or  a  surgical  needle  with  cutting  edge. 

When  using  such  surgical  needles  it  is  a  good  plan  to  sharpen  the  cutting  edge  on 
a  fine-grained  whetstone.  Afterward  the  needle  should  be  sterilized  by  boiling. 
Sterilization  of  a  needle  in  the  flame  blunts  the  cutting  edge.  A  steel  pen  with  one 
nib  broken  off  or  the  glass  needle  of  Wright  may  also  be  used.  To  make  a  glass 
needle,  pull  straight  apart  a  piece  of  capillary  tubing  in  a  very  small  flame.  Tap 
the  fine  point  to  break  off  the  very  delicate  extremity.  Scarcely  any  pain  attends 
the  use  of  such  a  needle.  In  puncturing  either  the  tip  of  the  finger  or  lobe  of  the 
ear  a  quick  piano-touch-like  stroke  should  be  used.  The  ear  is  preferable,  as  it  is 
less  sensitive  and  there  is  less  danger  of  infection.  Before  puncturing,  the  skin 
should  be  cleaned  with  70%  alcohol  and  allowed  to  dry.  It  is  advisable  to  sterilize 
the  needle  before  using  it. 


172  MICROMETRY   AND  BLOOD   PREPARATIONS 

The  first  drop  of  blood  which  exudes  should  be  taken  up  on  the  paper 
of  the  Tallquist  hsemoglobinometer,  using  subsequent  ones  for  the  blood 
pipettes  and  smears.  If  it  is  necessary  to  make  a  complete  blood  ex- 
amination, it  is  rather  difficult  to  draw  up  the  blood  in  the  pipettes, 
dilute  it,  and  then  get  material  for  fresh  blood  preparations  and  films 
without  undue  squeezing,  which  is  to  be  avoided.  Of  course,  fresh 
punctures  can  be  made.  Ordinarily,  complete  blood  examinations  are 
not  called  for.  It  is  only  a  white  count  or  a  differential  count  or 
an  examination  for  malaria  that  is  required. 

As  a  practical  point  it  is  very  rare  that  a  red  count  is  indicated.  There  is  one 
point  not  sufficiently  recognized  by  physicians  and  that  is  that  a  call  for  a  routine 
blood  examination  is  not  apt  to  be  as  carefully  conducted  as  one  calling  for  a  specific 
feature.  Without  disparaging  the  necessity  of  routine  examinations  of  urine  as 
well  as  blood  it  is  a  fact  that  the  internist  who  knows  what  he  wants  gets  better 
results  from  the  laboratory  man. 

HEMOGLOBIN  ESTIMATION. 

The  most  accurate  instrument  for  this  purpose  is  the  Miescher 
modification  of  the  v.  Fleischl  haemoglobinometer. 

The  magenta-stained  glass  wedge  for  comparison  with  the  diluted  blood  is  similar 
in  each  instrument,  but  by  the  use  of  a  diluting  pipette  accurate  dilutions  are  possible 
in  the  Miescher.  There  are  two  cells  provided — one  12  millimeters  high,  the  other 
15  millimeters;  the  idea  of  this  being  to  enable  one  to  make  separate  comparisons 
and  to  select  the  central  part  of  the  glass- wedge  scale,  where  comparison  is  more 
accurate  than  at  the  ends.  As  these  cells  contain  columns  of  diluted  blood  propor- 
tionately as  5  to  4,  we  should  have  similar  readings  when  we  multiply  the  reading 
on  the  scale  with  the  15  mm.  cell  by  4/5. 

The  mixing  pipette  is  graduated  with  the  marks  1/2,  2/3  and  i/i — the  first 
giving  a  dilution  of  i  to  400  (when  the  diluent,  3,0.1%  soda  solution,  is  drawn  up  to 
the  mark  above  the  bulb)  the  second  of  i  to  300  and  the  last  of  i  to  200. 

Artificial  light  preferably  from  a  candle  is  necessary.  There  is  a  table  accom- 
panying each  instrument  which  shows  the  value  for  that  particular  instrument  in 
milligrams  per  liter  of  haemoglobin  for  any  reading  obtained  on  the  scale. 

The  apparatus  is  expensive,  requires  considerable  time  and  care  in  the  making 
of  estimations,  and  is  exclusively  an  instrument  for  a  well-equipped  laboratory. 

Sahli's  Haemometer. — A  simple  and  apparently  very  scientific 
instrument  which  has  been  recently  introduced  is  the  Sahli  modifica- 
tion of  the  Gower  haemoglobinometer.  Instead  of  the  tinted  glass,  or 
gelatin  colored  with  picrocarmine  to  resemble  a  definite  blood  dilution, 
Sahli  uses  as  a  standard  the  same  coloring  matter  as  is  present  in  the  tube 
containing  the  blood.  By  acting  on  blood  with  ten  times  its  volume  of 


HEMOGLOBIN   ESTIMATION 


173 


N/io  HC1,  haematin  hydrochlorate  is  produced,  which  gives  a  brownish- 
yellow  color.  In  the  standard  tube,  which  is  sealed,  a  dilution  repre- 
senting i%  of  normal  blood  is  used.  To  apply  this  test,  pour  in  N/io 
HC1  to  the  mark  10  on  the  scale  of  the  graduated  tube.  Add  to  this  20 
cubic  millimeters  of  the  blood  to  be  examined,  drawn  up  by  the  capillary 
pipette  provided.  So  soon  as  the  mixture  assumes  a  clear  bright  dark 
brown  color,  add  water  drop  by  drop  until  the  color  of  the  tubes  matches. 
The  reading  of  the  height  of  the  aqueous  dilution  on  the  scale  gives 
the  Hb.  reading.  The  tubes  are  encased  in  a 
vulcanite  frame  with  rectangular  apertures. 
This  gives  the  same  optical  impression  as 
would  planoparallel  glass  sides. 

The  most  accurate  readings  are  obtained  with  ar- 
tificial light  in  a  dark  room  but  almost  as  satisfactory 
comparisons  can  be  obtained  with  natural  light  from 
a  window.  It  is  advisable  to  turn  the  ruled  side 
around  so  that  one  may  match  colors  without  being 
influenced  in  his  determination  by  the  scale. 

The  apparatus  must  be  kept  in  a  dark  place  as 
strong  light  will  change  the  color  of  the  standard 
tube.  It  is  recommended  that  the  N/io  HC1  be 
preserved  with  chloroform. 

Tallquist's  Haemoglobin  Scale. — This  is 
a  small  book  of  specially  prepared  filter-pa- 
per with  a  color-scale  plate  of  ten  shades  of 
blood  colors.  These  are  so  tinted  as  to  match 
blood  taken  up  on  a  piece  of  the  filter-paper 
and  are  graded  from  10  to  100.  So  soon  as  the 
blood  on  the  filter-paper  has  lost  its  humid 
gloss,  the  comparison  should  be  made.  This 
is  best  done  by  shifting  the  blood-stained 
piece  of  filter-paper  suddenly  from  one  to  the 
other  of  the  holes  cut  in  each  shade — -the 
piece  of  filter-paper  being  underneath  the  color 

plate.  At  least  a  square  centimeter  of  the  filter  paper  should  be  stained 
by  the  blood.  Daylight  coming  from  a  window  to  the  rear  or  at  the  side 
should  be  used  in  making  the  comparison.  The  error  with  this  method 
is  probably  not  over  10%  after  a  little  experience.  If  the  colored  plate 
is  not  kept  in  the  dark,  the  tints  tend  to  fade. 


FIG.    51. — Sahli's  haemo- 
globinometer.  (Greene.} 


174 


MICROMETRY   AND   BLOOD    PREPARATIONS 


To'  COUNT  BLOOD-CORPUSCLES. 

The  instrument  almost  universally  used  is  the  Thoma-Zeiss  haemacy- 
tometer.  The  apparatus  consists  of  two  pipettes,  one  for  leukocytes, 
graduated  to  give  a  dilution  of  i  to  10  or  greater;  the  other  for  red  cells 
to  give  a  dilution  of  a  i  to  i  oo  or  greater.  The  white  pipette  has  the  mark 
ii  above  the  bulb  and  the  red  pipette  the  mark  101.  In  addition,  there 
is  a  counting  chamber.  This  consists  of  a  square  of  glass  with  a  round 
hole  in  the  center.  Occupying  the  center  of  this  round  hole  is  a  circu- 
lar disc  of  glass  of  less  diameter,  SD  that  an  encircling  channel  is  left. 

The  square  and  the  circle  of  glass  are 
cemented  to  a  heavy  glass  slide.  The  sur- 
faces of  each  are  absolutely  level  and 
highly  polished.  That  of  the  circular  disc 
is  ruled  into  squares  of  varying  size  and  is 
exactly  i/io  of  a  millimeter  below  the 
level  of  the  surface  of  the  surrounding 
glass  square. 

When  a  polished  piano-parallel  cover- 
glass  rests  on  the  shelf,  as  the  outer  square 
glass  is  termed,  there  is  a  space  left  be- 
tween its  under  surface  and  the  ruled  disc 
of  o.  i  millimeter.  The  channel  around  the 
disc  is  termed  the  moat  or  ditch.  The  most 
desirable  rulings  are  those  of  Turck  and  of 
Zappert.  In  these  the  entire  ruled  surface 

consists  of  nine  large  squares,  each  i  millimeter  square.  These  are  sub- 
divided, and  in  the  central  large  square  are  to  be  found  the  small  squares 
used  for  averaging  the  red  cells.  These  small  squares  are  1/20  of  a  milli- 
meter square  and  are  arranged  in  nine  groups  of  sixteen  small  squares 
by  bordering  triple-ruled  lines.  As  the  unit  in  blood  counting  is  the 
cubic  millimeter,  if  one  counted  all  the  white  cells  lying  within  one  of 
the  large  squares  (i  millimeter  square),  he  would  have  only  counted  the 
cells  in  a  layer  i/io  of  the  required  depth,  so  that  it  would  be  necessary 
to  multiply  the  number  obtained  by  10.  This  product,  multiplied  by 
the  dilution  of  the  blood,  would  give  the  number  of  white  cells  in  a  cubic 
millimeter  of  undiluted  blood. 


FIG.  5  2. — Thomas- Zeiss  blood 
counter  showing  pipette,  count- 
ing chamber,  and  ruled  field. 
(Greene.) 


To  make  a  red  count:  Having  a  fairly  large  drop  of  blood,  apply  the  tip  of  the 
10 1  pipette  to  it  and,  holding  the  pipette  horizontally,  carefully  and  slowly  draw  up 


THE  COUNTING  OF  RED  CELLS  175 

with  suction  on  the  rubber  tube  a  column  of  blood  to  exactly  0.5  or  i.  The  variation 
of  1/25  of  an  inch  from  the  mark  would  make  a  difference  of  almost  3%.  If  the 
column  goes  above  0.5,  it  can  be  gently  tapped  down  on  a  piece  of  filter-paper  until 
the  0.5  line  is  cut.  Now  insert  the  tip  of  the  pipette  into  some  diluting  fluid  and, 
revolving  the  pipette  on  its  long  axis  while  filling  it  by  suction,  you  continue  until 
the  mark  101  is  reached.  A  variation  of  1/25  of  an  inch  at  this  mark  would  only 
give  an  error  of  about  1/30  of  i%.  After  mixing  thoroughly  by  shaking  for  one 
or  two  minutes,  the  fluid  in  the  pipette  below  the  bulb  is  expelled  (this,  of  course,  is 
only  diluting  fluid).  A  drop  of  the  diluted  blood  of  a  size  just  sufficient  to  cover 
the  disc  when  the  cover-glass  is  adjusted,  is  then  deposited  on  the  disc  and  the  cover- 
glass  applied  by  a  sort  of  sliding  movement,  best  obtained  by  using  a  forceps  in 
one  hand  assisted  by  the  thumb  and  index-finger  of  the  other. 
Among  diluting  fluids  Toisson's  is  probably  the  best: 

Sodium  chloride,  i  gram 

Sodium  sulphate,  8  grams 

Glycerine,  30  c.c. 

Distilled  water,  160  c.c. 

Dissolve  the  sodium  chloride  and  the  sodium  sulphate  in  the  glycerine  water  and 
add  sufficient  methyl  or  gentian  violet  to  give  a  rich  violet  tint. 

A  2  1/2%  solution  of  potassium  bichromate  makes  a  very  satisfactory  diluting 
fluid  in  the  counting  of  red  cells. 

A  salt  solution  of  about  i%  strength,  tinged  with  about  i  drop  of  a  saturated 
alcoholic  solution  of  gentian  violet  to  about  50  c.c.,  is  a  good  substitute,  or  the  salt 
solution  alone  will"  answer  when  no  white  count  is  to  be  made  at  the  same  time  as  the 
red  one. 

It  is  important  to  work  quickly  in  adjusting  the  cover-glass,  or  there  will  be  cells 
settling  in  the  center  of  the  drop  from  a  greater  depth  than  the  one  which  the  apposi- 
tion of  the  cover-glass  makes  (i/io  millimeter  deep). 

A  good  preparation  should  show: 

1.  Presence  of  Newton's  rings. 

2.  Absence  of  air  bubbles. 

3.  Entire  surface  of  ruled  disc  covered. 

4.  Equal  distribution  of  cells. 

Before  counting,  about  five  minutes  should  be  allowed  for  the  set- 
tling of  the  cells. 

It  will  be  remembered  that  the  small  squares  are  1/20  millimeter  square.  The 
depth  of  fluid  from  upper  surface  of  shelf  to  lower  surface  of  cover-glass  is  i/io  mm. 
Hence  each  space  embraced  by  the  small  square  and  the  depth  of  fluid  is  1/4000  of 
the  unit  used  in  estimating  number  of  corpuscles  in  blood,  or  i  cubic  millimeter 
(1/20X1/20X1/10=1/4000).  Count  100  of  the  small  squares  .(this  enables  one 
to  use  decimals).  There  are  nine  squares  between  triple-ruled  lines,  each  containing 
sixteen  small  squares.  Count  the  number  of  corpuscles  in  the  sixteen  small  squares 
contained  in  upper  left-hand  triple-ruled  square.  Put  down  this  count.  Next 


176  MICROMETRY    AND   BLOOD    PREPARATIONS 

count  corpuscles  in  the  adjoining  sixteen  squares.  Put  down  this  count.  Then  in 
third  sixteen  squares.  Put  down  the  number.  Now  move  down  to  next  row  of 
three  triple-ruled  squares.  Count  the  number  of  corpuscles  in  each  of  the  three 
sixteen-square  spaces  and  set  down  the  numbers  for  addition.  We  have  now  counted 
ninety-six  small  squares  (6X 16).  Count  at  any  place  four  additional  small  squares 
and  add  number  of  blood-cells  contained  therein  to  those  in  the  ninety-six  small 
squares  already  counted.  Divide  the  sum  by  100  or  simply  point  off  two  decimals. 
This  gives  the  average  for  each  small  square.  Multiply  this  by  the  dilution  and  then 
(as  the  small  square  is  only  1/4000  cu.  mm.)  by  4000.  This  will  give  the  number  of 
corpuscles  in  i  cubic  millimeter.  Example:  100  small  squares  contained  655  red 
cells.  Pointing  off,  6.55  equals  average  number  of  red  cells  per  small  square. 
Multiply  by  dilution  (200)  and  then  by  4000  (the  small  square  is  4000  times  smaller 
than  the  unit:  i  cu.  mm.) — 6.55X200=1310X4000  =  5,240,000. 

At  least  100  small  squares,  and  preferably  200  should  be  counted. 
If  the  blood  appears  normal,  one  may  simply  count  the  number  of  red 
cells  in  five  of  the  sixteen  small  square  spaces  (eighty  small  squares). 
Having  added  the  numbers  and  multiplying  by  10,000,  you  obtain  the 
number  of  cells  in  i  cubic  millimeter.  (Eighty  small  squares  is  1/50  of 
the  unit  of  i  cu.  mm.,  or  4000  small  squares.  The  blood  dilution  being 
i  to  200,  we  have  50 X  200  X number  of  cells  in  eighty  small  squares.) 

In  counting,  count  corpuscles  lying  on  the  lires  above  and  to  the  right.  Do  not 
count  those  lying  on  lines  below  and  to  the  left. 

In  the  small  squares  count  only  corpuscles  lying  in  the  space  or  cutting  the  upper 
line.  This  prevents  counting  the  same  cell  twice. 

To  Count  White  Cells.— Draw  up  the  fluid  in  the  white  pipette  to 
the  mark  0.5.  Then,  still  holding  the  pipette  as  near  the  horizontal  as 
possible,  because  the  column  of  blood  tends  to  fall  down  in  the  larger 
bore,  draw  up  by  suction  a  diluting  fluid  which  will  disintegrate  the  red 
cells  without  injuring  the  whites.  The  best  fluid  is  0.3  %  of  glacial  acetic 
acid  in  water.  This  makes  the  white  cells  stand  out  as  highly  refractile 
bodies.  Some  prefer  to  tinge  the  fluid  with  gentian  violet.  The  0.5 
mark  is  preferred  because  it  takes  a  very  large  drop  of  blood  to  fill  the 
tube  up  to  the  i  mark  and  if  there  is  much  of  a  leukocytosis  a  i  to  10 
dilution  is  not  sufficient.  In  leukaemic  blood  it  is  better  to  use  the  red 
pipette  with  the  0.3%  acetic  acid  solution. 

The  blood  having  been  drawn  up  to  0.5,  we  have  a  dilution  of  i  to  20. 
Making  a  preparation,  exactly  as  was  done  in  the  case  of  the  red  count, 
we  count  all  of  the  white  cells  in  one  of  the  large  squares  (i  sq.  mm.). 
The  cross  ruling  greatly  facilitates  this.  Note  the  number.  Then 
count  a  second  and  a  third  large  square.  Strike  an  average  for  the  large 
squares  counted  and  multiply  this  by  10,  as  the  depth  of  the  fluid  gives 


THE    COUNTING    OF    WHITE    CELLS  177 

a  content  equal  to  only  i/io  of  a  cubic  millimeter.  Then  multiply 
by  the  dilution.  Example:  First  large  square  50;  second  large  square 
70;  third  large  square  60.  Average  60.  Then  60X10X20  =  12,000, 
the  number  of  leukocytes  in  i  cubic  millimeter  of  blood.  The  count 
may  be  made  with  a  low  power  (2/3 -inch  objective)  as  the  leukocytes 
stand  out  like  pearls.  It  is  better,  however,  to  use  a  higher  power,  so 
that  pieces  of  foreign  material  may  be  recognized  and  not  enumerated 
as  white  cells. 

When  it  is  desired  to  make  a  white  count  with  the  same  preparation  as  is  used  for 
the  red  one,  especially  if  the  ruling  is  of  the  old  style  (only  central  ruling  and  not  in 
nine  large  squares  as  with  Zappert  and  Tiirck),  it  is  advisable  to  make  use  of  the 
method  of  counting  by  fields.  With  a  Leitz  No.  4  ocular  and  a  No.  6  objective,  with 
a  tube  length  of  120  millimeters,  it  will  be  observed  that  the  field  so  obtained  has  a 
diameter  of  eight  small  squares.  Now,  remembering  that  the  area  of  a  circle  equals 
the  square  of  the  radius  multiplied  by  TT,  or  3.1416,  we  have  the  following  calculation: 
The  diameter  being  eight  small  squares,  the  radius  would  be  four  small  squares. 
Squaring  the  radius,  we  have  sixteen.  This  multiplied  by  3.1416  gives  us  fifty. 
This  means  that  every  field,  with  the  microscope  adjusted  as  stated,  contains  fifty 
of  the  small  squares,  or  1/80  of  the  unit  of  one  cubic  millimeter  of  the  diluted  blood. 

By  keeping  a  single  red  cell  in  view  while  moving  the  mechanical  stage  from  right 
to  left  or  from  above  downward,  we  know  that  a  new  field  of  fifty  small  squares  is 
brought  into  view  when  the  circumference  of  the  field  cuts  this  individual  cell. 
Example:  As  2000  small  squares  would  ordinarily  be  a  sufficient  number  to  count 
for  a  white  count,  this  would  require  us  to  count  the  number  of  leukocytes  in  forty 
of  the  designated  microscopic  fields  (this,  of  course,  is  only  one-half  the  unit,  hence 
we  should  multiply  by  2).  Counted  forty  fields  and  noted  fifty  white  cells.  50X2 
=  100X200  (the  dilution  in  red  pipette)  =  20,000.  Consequently  20,000  would 
represent  the  number  of  leukocytes  in  one  cubic  millimeter  of  the  blood  examined. 

After  making  a  blood  count,  the  hsemacytometer  slide  should  be  cleaned  with  soap 
and  water  and  then  rubbed  dry,  preferably  with  an  old  piece  of  linen.  As  the 
accuracy  of  the  counting  chamber  depends  upon  the  integrity  of  the  cement,  any 
reagent  such  as  alcohol,  xylol,  etc.,  and,  in  particular,  heat,  will  ruin  the  instrument. 
The  pipettes  should  be  cleaned  by  inserting  the  ends  into  the  tube  from  a  vacuum 
pump,  as  a  Chapman  pump.  First  draw  water  or  i%  sod.  carbonate  solution 
through  the  pipette,  then  alcohol,  then  ether,  and  finally  allow  air  to  pass  through 
to  dry  the  interior.  If  the  interior  is  stained,  use  i%  HC1  in  alcohol.  If  a  vacuum 
pump  is  not  at  hand,  a  bicycle  pump  or  suction  by  mouth  will  answer. 

PREPARATIONS  FOR  THE  STUDY  OF  FRESH  BLOOD. 

Many  authorities  prefer  a  fresh-blood  specimen  to  a  stained  dried 
smear  in  the  study  of  parasites  of  the  blood.  In  malaria  in  particular 
there  is  so  much  information  as  to  species  to  be  obtained  from  a  fresh 
specimen  that  the  employment  of  this  method  should  never  be  neglected. 


178  'MICROMETRY   AND   BLOOD    PREPARATIONS 

While  waiting  for  the  film  to  stain  one  has  five  or  six  minutes  which 
could  not  be  better  spent  then  in  examining  the  fresh  specimen  which 
only  requires  a  moment  to  make. 

Manson's  Method. — -Have  a  perfectly  clean  cover-glass  and  slide.  Touch  the 
apex  of  the  exuding  drop  of  blood  with  the  cover-glass  and  drop  it  on  the  center  of 
the  slide.  The  blood  flows  out  in  a  film  which  exhibits  an  "empty  zone"  in  the 
center.  Surrounding  this  we  have  the  "zone  of  scattered  corpuscles,"  next  the 
"single  layer  zone"  and  the  "zone  of  rouleaux"  at  the  periphery.  It  is  well  to  ring 
the  preparation  with  vaseline.  When  desiring  to  demonstrate  the  flagellated  bodies 
in  malaria,  it  is  well  to  breathe  on  the  cover-glass  just  prior  to  touching  the  drop  of 
blood. 

The  Method  of  Ross  is  very  easy  of  application  and  gives  most  satisfactory 
preparations.  Take  a  perfectly  clean  slide,  and  make  a  vaseline  ring  or  square  of 
the  size  of  the  cover-glass.  Then,  having  taken  up  the  blood  on  the  cover-glass, 
drop  it  so  that  its  margin  rests  on  the  vaseline  ring.  Gently  pressing  down  the  cover- 
glass  on  the  vaseline  makes  beautiful  preparations  which  keep  for  a  very  long  time. 
If  it  is  desired  to  study  the  action  of  stains  on  living  cells,  this  method  is  also  appli- 
cable. A  very  practical  way  to  do  this  is  to  tinge  0.85%  salt  solution  containing  i% 
sodium  citrate  (the  same  as  is  used  in  opsonic  work)  with  methylene  azur,  gentian 
violet,  or  methyl  green.  With  a  Wright  bulb  pipette,  take  up  one  part  of  blood, 
then  one  part  of  tinted  salt  solution.  Mix  them  quickly  on  a  slide  and  then  deposit 
a  small  drop  of  the  mixture  in  the  center  of  the  vaseline  ring  and  immediately  apply 
a  cover-glass  and  press  down  the  margins  as  before.  This  method  will  be  found  of 
great  practical  value. 

A  METHOD  FOR  MAKING  DIFFERENTIAL  LEUKOCYTE  COUNT  IN  SAME 
PREPARATION  AS  FOR  WHLJE  COUNT. 

Employ  the  same  technic  as  in  making  the  ordinary  white  count 
but  using  as  a  diluting  fluid  a  2  %  formalin  solution  to  which  has  been 
added  one  drop  of  Giemsa's  stain  for  each  c.c.  just  before  making  the 
blood  examination. 

The  best  results  are  obtained  when  the  mixing  in  the  pipette  bulb  is  done  im- 
mediately after  taking  up  the  blood  and  diluent.  Recently  I  have  found  it  necessary 
to  add  enough  N/i  NaOH  to  the  commercial  formalin  to  bring  it  to  +i.  Of  this 
I  use  i  1/2%  in  a  1/2%  glycerine  solution  instead  of  water. 

The  usual  technic  in  making  the  haemocytometer  preparation  is  employed- 
using  a  Tiirck  ruling.  Count  the  leukocytes  in  the  three  upper  or  lower  i  sq.  mm. 
squares,  divide  by  3  to  obtain  an  average  per  sq.  mm.,  multiply  by  10  for  the  con- 
tent of  a  cubic  millimeter  and  then  by  20  for  the  dilution.  (Blood  to  0.5;  diluent 
to  IT.)  This  can  be  done  mentally  and  requires  no  calculation  on  paper.  Having 
counted  the  leukocytes,  again  go  over  the  same  portion  of  the  ruled  surface  and  count 
the  polymorphonuclears  and  estimate  the  percentage  of  these  to  the  total  leuko- 
cytes. The  majority  of  disrupted  cells  in  a  dry-stained  preparation  are  transitionals 
hence  the  percentage  of  polymorphonuclears  by  this  method  is  lower. 


STAINING    OF  BLOOD  179 

It  is  unnecessary  in  such  a  count  to  have  an  assistant;  of  course,  in  making  a 
complete  differential  count  it  is  preferable  to  have  some  one  tabulate  or  labor- 
iously to  do  this  one's  self. 

The  red  cells  are  practically  diaphanous  and  not  disintegrated  as  when  acetic 
acid  is  used  as  a  diluent,  consequently  it  is  easy  to  make  out  the  particular  red  cell 
as  to  size,  etc.,  containing  a  malarial  parasite. 

The  best  results  are  obtained  with  a  i/6-in  objective.  Higher  powers  are  of 
course  impracticable  by  reason  of  the  thickness  of  the  cover-glass  of  the  haemocy- 
tometer. 

The  following  are  the  appearances  of  the  various  leukocytes. 

Eosinophiles. — In  these  the  bilobed  nucleus  stains  rather  faintly  and  the  color 
is  greenish  blue.  The  eosinophile  granules  show  easily  as  coarse,  brickdust-colored 
particles. 

Polymorphonuclears. — The  nucleus  stains  a  deep,  rich,  pure  violet  but  less  in- 
tense than  that  of  the  small  lymphocyte.  The  shape  of  the  nucleus  is  typically 
three  or  four  lobed  but  even  when  of  the  horseshoe  shape  of  a  transitional  nucleus 
is  easily  recognizable  by  the  intensity  of  the  violet  staining.  That  which  makes  the 
polymorphonuclears  very  easy  of  differentiation  is  the  distinctness  of  the  cell  out- 
lines produced  by  the  fine  yellowish  granulations  in  the  cytoplasm. 

Small  Lymphocytes. — The  nucleus  is  perfectly  round  and  stains  a  deep  violet. 
It  is  almost  impossible  to  make  out  any  cytoplasmic  fringe. 

Large  Lymphocytes. — The  nucleus  here  is  round  and  of  a  lighter  violet  than  that 
of  the  small  lymphocyte.  The  cytoplasm  is  blue,  nongranular,  and  sharply  defined 
from  the  nucleus. 

Large  Mononuclears. — These  show  a  washed-out,  slate-colored  nucleus  which 
blends  with  the  gray  slate-blue  staining  of  the  cytoplasm  so  that  there  is  an  in- 
definiteness  of  outline  in  the  more  or  less  irregularly  contoured  nucleus. 

Transitionals. — These  show  the  same  characteristics  as  the  large  mononuclears, 
but  with  a  more  faintly  stained  and  more  indented  nucleus.  The  large  mono- 
nuclears and  transitionals  stand  out  as  slate-colored  cells.  When  very  much  degen- 
erated these  cells  have  a  greenish  hue. 

Mast  Cells. — The  granulations  show  as  a  rich  maroon  or  reddish-violet  color. 

The  young  ring  forms  of  malaria  show  as  violet-blue  areas  in  the  red  cells.  When 
half-grown  or  approaching  the  merocyte  stage,  the  containing  red  cell  takes  on  a 
faint  pink  coloration,  thereby  differentiating  it  from  the  noninfected  red  cells.  At 
the  same  time  the  parasite  is  extruded  and  has  the  appearance  of  a  violet-blue  body 
projecting  from  the  margin  of  the  red  cell.  It  is  as  if  a  blue  body  were  budding 
from  a  pink  one. 

It  is  an  easy  matter  with  this  method  to  count  the  number  of  trypanosomes  or 
malarial  crescents  in  a  cubic  millimeter  of  blood. 


PREPARATION  AND  STAINING  OF  DRIED  FILMS. 

When  preparations  are  desired  for  a  differential  count,  Ehrlich's 
method  of  making  films  is  to  be  preferred,  as  the  different  types  of  leuko- 
cytes are  more  evenly  distributed.  In  making  smears  by  spreading, 


i8o 


MICROMETRY   AND  BLOOD   PREPARATIONS 


there  is  a  tendency  for  the  polymorphonuclears  to  be  concentrated  at 
the  margin  while  lymphocytes  remain  in  the  central  part  of  the  film. 

In  Ehrlich's  method  we  have  perfectly  clean  dry  cover-slips.  Take 
up  a  small  drop  of  blood  without  touching  the  surface  of  the  ear  or  finger. 
Drop  this  cover-glass  'immediately  on  a  second  one  and  as  soon  as  the 
blood  runs  out  in  a  film,  draw  the  two  cover-slips  apart  in  a  plane  par- 
allel to  the  cover-glasses.  Slide  them  apart.  Ehrlich  uses  forceps  to 
hold  the  cover-glasses  to  avoid  moisture  from  the  fingers. 

Of  the  various  methods  of  spreading  films  on  slides  there  is  none 


FIG.  53. — Blood  technic.  i,  2,  3,  Method  for  making  blood  smear  on  slide;  4, 
U  tube  for  resting  slides  while  staining;  5,  slide  showing  grease  pencil  marking, 
marking  prevents  stain  from  overflowing;  6,  method  for  drawing  apart  cover-glasses 
in  making  blood  smear. 

equal  to  that  described  by  Daniels.  In  this  the  drop  of  blood  is  drawn 
along  and  not  pushed  along.  The  films  are  even,  can  be  made  of  any 
desired  thickness  by  changing  the  angle  of  the  drawing  slide,  and  there 
is  little  liability  of  crushing  pathological  cells.  Take  a  small  drop  of 
blood  on  the  end  of  a  clean  slide.  Touch  a  second  slide  about  1/2 
inch  from  end  with  the  drop  and  as  soon  as  the  blood  runs  out  along 
the  line  of  the  slide  end,  slide  it  at  an  angle  of  45°  to  the  other  end  of  the 


BLOOD    SMEARS  l8l 

horizontal  slide.  The  blood  is  pulled  or  drawn  behind  the  advancing 
edge  of  the  advancing  slide.  An  angle  less  than  45°  makes  a  thinner 
film;  one  greater,  a  thicker  film. 

Instead  of  a  slide  a  square  cover-glass  may  be  used  and  if  the  edge  be  smooth  it 
makes  a  more  satisfactory  spreader  than  the  slide.  Many  workers  prefer  the  Ross 
thick-film  method  in  examining  for  malaria.  In  this  about  one-half  of  a  drop  of 
blood  is  smeared  out  over  a  surface  about  equal  to  that  of  a  square  cover-glass  and 
allowed  to  dry.  It  is  then  flooded  with  a  0.1%  aqueous  solution  of  eosin  for 
about  15  minutes.  The  preparation  is  then  gently  washed  with  water  and  then 
treated  with  a  polychrome  methylene-blue  solution.  After  a  few  seconds  this  is 
carefully  washed  off  and  the  preparation  dried  and  examined. 

Instead  of  the  Daniels  method  some  prefer  to  take  up  the  drop  of  blood  on  the 
slide  on  which  the  smear  is  to  be  made,  about  1/2  inch  from  the  end.  Then  apply 
the  spreader  slide  and  so  soon  as  the  drop  runs  along  the  end  of  the  spreader  slide 
proceed  as  above  described. 

Of  the  various  methods  of  making  smears  by  means  of  cigarette  paper,  rubber 
tissue,  needles,  etc.,  the  best  seems  to  be  to  take  a  piece  of  capillary  glass  tubing  and 
use  this  instead  of  a  needle  in  making  the  film.  There  is  one  advantage  about  the 
strip  of  cigarette  paper  touched  to  the  drop  of  blood  and  drawn  out  along  the  slide 
or  cover-glass,  and  that  is  that  it  is  almost  impossible  not  to  make  a  working  prepa- 
ration by  this  method. 

In  the  making  of  smears  the  chief  points  are  to  make  the  smears  as 
soon  after  taking  the  blood  as  possible  and  to  have  slides  and  cover- 
glasses  scrupulously  clean.  It  is  well  to  flame  all  slides  and  cover- 
glasses  which  are  to  be  used  for  blood-work.  This  is  the  best  method 
of  getting  rid  of  grease. 

Fixation  of  Film. — In  Wright's,  Leishman's,  and  other  similar  stains  the  methyl- 
alcohol  solvent  causes  the  fixation.  In  staining  with  Giemsa's  stain,  Ehrlich's 
tri-acid,  hsemotoxylin  and  eosin,  Smith's  formol  fuchsin,  and  with  thionin,  separate 
fixation  is  necessary.  For  Giemsa  and  thionin,  either  absolute  alcohol  (ten  to 
fifteen  minutes),  or  methyl  alcohol  (two  to  five  minutes)  answer  well. 

Formalin  vapor,  for  five  to  ten  seconds,  is  also  used  for  fixation.  For  Ehrlich's 
tri-acid,  haemotoxylin  and  eosin  and  formol  fuchsin,  heat  gives  the  best  results. 
The  best  method  is  to  place  the  films  in  an  oven  provided  with  a  thermometer. 
Raise  the  temperature  of  the  oven  to  135°  C.  and  then  remove  the  burner.  After 
the  oven  has  cooled,  take  out  the  fixed  slides  or  slips. 

Some  prefer  to  place  a  crystal  of  urea  on  the  slide,  then  hold  it  over  the  flame 
until  the  urea  melts.  This  shows  that  a  temperature  between  130°  and  135°  C.  has 
been  reached. 

One  of  the  handiest  methods  is  to  drop  a  few  drops  of  95%  alcohol  on  the  slide 
or  cover-glass.  Allow  this  to  flow  over  the  entire  surface;  then  get  rid  of  the  excess 
of  alcohol  by  touching  the  edge  to  a  piece  of  filter-paper  for  a  second  or  two.  Then 
light  the  remaining  alcohol  film  from  the  flame  and  allow  the  burning  alcohol  to 
burn  itself  out.  A  chemical  fixation  which  gives  good  fixation  for  haematoxylin 


182  MICROMETRY    AND   BLOOD    PREPARATIONS 

and  tri-acid  stains  (not  equal  to  heat)  is  a  modification  of  Zenker's  fluid  (Whitney). 
To  Muller's  fluid,  which  is  potassium  bichromate  2  grams,  sodium  sulphate  i  gram, 
and  water  100  c.c.,  add  5  grams  of  bichloride  of  mercury  and  5  c.c.  of  nitric  acid 
(C.  P.).  Fixation  is  obtained  in  five  seconds. 

When  using  corrosive  sublimate  fixation  one  should  after  thorough  washing  in 
water  treat  the  film  with  Gram's  iodine  solution  for  about  two  minutes  and  then  wash 
with  70%  alcohol  until  the  yellow  color  of  the  film  disappears.  Methyl  alcohol 
for  two  minutes  is  satisfactory. 

Staining  Blood-films. — As  separate  staining  with  eosin  and  methyl- 
ene  blue  rarely  gives  good  preparations  and  as  the  modifications  of  the 
Romanowsky  stain  recommended  are  easy  to  make  and  employ,  and 
give  much  greater  information,  the  separate  method  of  staining  is  not 
recommended.  The  most  satisfactory  single  stain  is  thionin. 

Rees'  Thionin  Solution. — Take  of  thionin  1.5  gram,  alcohol  10  c.c.,  aqueous 
solution  of  carbolic  acid  (5%)  100  c.c.  Keep  this  as  a  stock  solution.  It  should 
be  at  least  two  weeks  old  before  using.  For  use,  filter  off  5  c.c.  and  make  up  to 
20  c.c.  with  water. 

1.  Fix  films  (a)  by  heat,  (b)  by  alcohol  and  ether,  or  (c)  preferably  by    i% 
formalin  in  95%  alcohol  for  one  minute. 

2.  Stain  for  from  ten  to  twenty  minutes.     Wash  and  mount.     Malarial  parasites 
are  stained  purplish;  nuclei  of  leukocytes,  blue;  red  cells,  faint  greenish-blue. 

Ehrlich's  Triacid  or  Triple  Stain. — There  are  required: 

1.  Sat.  aq.  sol.  orange  G.     (Dissolve  3  grams  in  50  c.c.  water.) 

2.  Sat.  aq.  sol.  acid  fuchsin.     (Dissolve  10  grams  in  50  c.c.  water.) 

3.  Sat.  aq.  sol.  methyl  green.     (Dissolve  10  grams  in  50  c.c.  water.) 

These  three  solutions  may  be  kept  as  stock  solutions.  They  keep  well  in  the  dark. 
To  make  the  stain,  add  9  c.c.  of  No.  2  (acid  fuchsin)  to  18  c.c.  of  No.  i  (orange  G.). 
After  they  are  mixed  thoroughly,  add  20  c.c.  of  No.  3  (methyl  green).  Then  after 
the  first  3  ingredients  are  well  mixed,  add  5  c.c.  of  glycerin.  Mix,  then  add  15  c.c. 
of  alcohol;  again  mix,  and  finally  add  30  c.c.  of  distilled  water.  Keep  the  mixed 
stain  about  one  week  before  using.  The  best  fixatives  are  heat  and  Whitneys' 
modified  Zenker.  To  use,  stain  films  from  two  to  five  minutes;  then  wash  and 
mount.  The  triacid  stain  is  a  good  tissue  stain.  The  objections  to  the  tri-acid 
stain  are  that  it  does  not  stain  malarial  parasites  or  mast  cells  and  that  failure  to 
obtain  good  results  is  of  frequent  occurrence. 

Wright's  Method. — The  stain  is  made  by  adding  i  gram  of  methylene  blue  (Grubler) 
to  100  c.c.  of  a  1/2%  solution  of  sodium  bicarbonate  in  water.  This  mixture  is 
heated  for  i  hour  in  an  Arnold  sterilizer.  The  flask  containing  the  alkaline  methyl  - 
ene-blue  solution  should  be  of  such  size  and  shape  that  the  depth  of  the  fluid  does 
not  exceed  2  1/2  inches.  When  cool,  add  to  the  methylene-blue  solution  500  c.c. 
of  a  i  to  1000  eosin  solution  (yellow  eosin,  water  soluble).  Add  the  eosin  solution 
slowly,  stirring  constantly  until  the  blue  color  is  lost  and  the  mixture  becomes 
purple  with  a  yellow  metallic  luster  on  the  surface,  and  there  is  formed  a  finely 
granular  black  precipitate.  Collect  this  precipitate  on  a  filter-paper  and  when 
thoroughly  dry  (dry  in  the  incubator  at  38°  C.)  dissolve  0.3  gram  in  100  c.c.  of  pure 


ROMANO WSKY   STAINING   METHODS  183 

methyl  alcohol  (acetone  free).  Wright  lately  has  recommended  using  o.  i  in  60  c.c. 
methyl  alcohol.  This  constitutes  the  stock  solution.  For  use  filter  off  20  c.c.  and 
add  to  the  filtrate  5  c.c.  of  methyl  alcohol. 

A  modification  by  Batch  is  very  satisfactory.  In  this  method  instead  of  poly- 
chroming  the  methylene  blue  with  sodium  bicarbonate  and  heat,  the  method  of 
Borrel  is  used.  Dissolve  i  gram  of  methylene  blue  in  100  c.c.  of  distilled  water. 
Next  dissolve  0.5  gram  of  silver  nitrate  in  50  c.c.  of  distilled  water.  To  the  silver 
solution  add  a  2  to  5%  caustic  soda  solution  until  the  silver  oxide  is  completely 
precipitated.  Wash  the  precipitated  silver  oxide  several  times  with  distilled  water. 
This  is  best  accomplished  by  pouring  the  wash-water  on  the  heavy  black  precipitate 
in  the  flask,  agitating,  then  decanting  and  again  pouring  on  water.  After  removing 
all  excess  of  alkali  by  repeated  washings,  add  the  methylene-blue  solution  to  the 
precipitated  silver  oxide  in  the  flask.  Allow  to  stand  about  ten  days,  occasionally 
shaking  until  a  purplish  color  develops.  The  process  may  be  hastened  in  an  incu- 
bator. When  polychroming  is  complete,  filter  off  and  add  to  the  nitrate  the  i  to 
1000  eosin  solution  and  proceed  exactly  as  with  Wright's  stain. 

In  Leishman's  method  the  polychroming  is  accomplished  by  adding  i  gram  of 
methylene  blue  to  100  c.c.  of  a  1/2%  solution  of  sodium  carbonate.  This  is  kept  at 
65°  C.  for  twelve  hours  and  allowed  to  stand  at  room  temperature  for  ten  days 
before  the  eosin  solution  is  added.  The  succeeding  steps  are  as  for  Wright's  stain. 

In  all  Romanowsky  methods  distilled  water  should  be  used.  If  not  obtainable, 
the  best  substitute  is  rain-water  collected  in  the  open  and  not  from  a  roof. 

Method  of  staining: 

1.  Make  films  and  air  dry. 

2.  Cover  dry  film  preparation  with  the  methyl-alcohol  stain  for  one  minute 
(to  fix). 

3.  Add  water  to  the  stain  on  the  cover-glass  or  slide,  drop  by  drop,  until  a 
yellow  metallic  scum  begins  to  form.     It  is  advisable  to  add  the  drops  of  water 
rapidly  in  order  to  eliminate  precipitates  on  the  stained  film.     Practically,  we  may 
add  i  drop  of  water  for  every  drop  of  stain  used. 

4.  Wash  thoroughly  in  water  until  the  film  has  a  pinkish  tint. 

5.  Dry  with  filter-paper  and  mount. 

Red  cells  are  stained  orange  to  pink;  nuclei,  shades  of  violet;  eosinophile  granules, 
red;  neutrophile  granules,  yellow  to  lilac;  blood  platelets,  purplish;  malarial  parasites, 
blue;  chromatin,  metallic-red  to  rose-pink 

Giemsa's  Modification  of  the  Romanowsky  Method. — This  is  one  of 
the  most  perfect  of  the  modifications.  The  objection  is  that  greater 
time  in  staining  films  is  required  than  with  the  Wright  or  Leishman 
method  and  the  stain  is  very  expensive. 

Take  of  Azur  II  eosin  0.3  gram.     Azur  II  0.08  gram. 

Dissolve  this  amount  of  dry  powder  in  25  c.c.  of  glycerine  at  60°  C. 
Then  add  25  c.c.  of  methyl -alcohol  at  the  same  temperature.  Allow 
the  glycerine  methyl-alcohol  solution  to  stand  overnight  and  then  filter. 
This  is  the  stock  stain.  To  use:  Dilute  i  c.c.  with  10  to  15  c.c.  of  dis- 


184  MICROMETRY   AND   BLOOD    PREPARATIONS 

tilled  water.     If  i  to  1000  potassium  carbonate  solution  is  used  instead 
of  water  it  stains  more  deeply. 

The  alkaline  diluent  is  used  to  obtain  the  course  stippling  in  malig- 
nant tertian  (Maurer's  clefts).  Having  fixed  the  smear  with  methyl 
alcohol  for  one  to  five  minutes,  pour  on  the  diluted  stain,  and  after 
fifteen  to  thirty  minutes  wash  off  and  continue  washing  with  distilled 
water  until  the  film  has  a  slight  pink  tinge.  For  Treponema  pallidum 
stain  from  two  to  twelve  hours. 

While  the  Romanowsky  methods  are  more  satisfactory  for  differential  counts  and 
for  the  demonstration  of  the  malarial  parasites,  and  especially  for  differentiating 
species,  yet  by  reason  of  the  liability  to  deterioration  in  the  tropics  of  methylene 
blue  the  hsematoxylin  methods  may  be  preferable.  Many  workers  in  blood-work 
and  cytodiagnosis  prefer  the  haematoxylin. 

1.  Fix  the  film  either  by  heat  with  methyl  alcohol  for  two  minuetes  or  with  Whit- 
ney's fixative.     Heat  is  to  be  preferred. 

2.  Stain  with  Meyer's  hemalum  or  Delafield's  haematoxylin  for  from  five  to 
fifteen  minutes  according    to  the  stain.     Frequently  three  minutes  will  be 
found  sufficient.     To  make  the  hemalum,  dissolve  0.5  gram  of  hae matin  in  25 
c.c.  of  95%  alcohol.     Next  dissolve  25  grams  of  ammonia  alum  in  500  c.c.  of 
distilled  water.     Mix  the  two  solutions  and  allow  to  ripen  for  a  few  days.     The 
stain  should  be  satisfactory  in  two  or  three  days. 

To  make  Delafield's  haematoxylin,  dissolve  i  gram  of  haematoxylin  crystals  in 
6  c.c.  of  95%  alcohol.  Add  this  to  100  c.c.  of  saturated  aqueous  solution  of 
ammonia  alum.  After  exposure  to  light  for  a  week,  the  color  changes  to  a 
deep  blue-purple.  Add  to  this  ripened  stain  25  c.c.  of  glycerine  and  25  c.c. 
of  methyl-alcohol  and,  after  it  has  stood  for  about  two  days,  filter.  The  stain 
should  be  filtered  from  time  to  time  as  a  sediment  forms.  This  makes  a  stock 
solution  which  should  be  diluted  10  to  15  times  with  water  when  staining. 

Mink's  Modification  of  TJnna's  Haematoxylin. 

Haematoxylin,  i  gram. 

Alum,  8  grams. 

Sulphur  (sublimed),  i  gram. 

Glycerine,  30  c.  c 

Alcohol,  50  c.c.. 

Water,  100  c.c. 

Dissolve  the  hsematoxylin  in  the  glycerine  in  a  mortar.  Dissolve  the  alum  in  the 
water  and  add  it  to  the  glycerine  haematoxylin  in  the  mortar.  Then  add  the  sulphur 
and  the  alcohol.  The  solution  ripens  in  about  three  to  four  days.  Allow  the  sedi- 
ment to  remain  in  the  bottom  of  the  bottle  containing  the  stain  and  filter  off  small 
quantities  as  needed. 

3.  Wash  for  two  to  five  minutes  in  tap  water  to  develop  the  haematoxylin  color. 

4.  Stain  either  with  a  i  to  1000  aqueous  solution  of  eosin  or  with  a  1/2  of  i% 


COAGULATION  RATE  OF  BLOOD  185 

eosin  solution  in  70%  alcohol.     The  eosin  staining  only  requires  fifteen   to 
thirty  seconds. 
5.  Wash  and  examine. 


lODOPHILIA. 

This  reaction  is  supposed  to  be  due  to  the  presence  of  glycogen, 
especially  in  the  polymorphonuclears,  in  suppurative  conditions. 

It  has  been  stated  that  a  differentiation  between  the  joint  involvement  in  gonor- 
rhoeal  infection  and  in  articular  rheumatism  may  be  made  from  iodophilia  being 
present  in  the  gonococcus  infection. 

Make  blood-smears  on  cover-glasses  as  usual,  and  after  they  dry,  but  without 
fixation,  mount  them  in  a  drop  of  the  following  solution: 

Iodine,  i  part. 

Potassium  iodide,  3  parts. 

Gum  arabic,  50  parts. 

Water,  100  parts. 

Small  brown  masses  in  the  polymorphonuclears  indicate  a  positive  iodophilia. 

Viscosity  of  the  Blood. — This  is  estimated  by  observing  the  relative  height  to 
which  blood  rises  in  capillary  tubes  as  compared  with  water,  and  normally  varies 
from  three  to  five.  The  higher  the  haemoglobin  content  the  greater  the  viscosity. 
Viscosity  is  high  in  arterio-sclerosis  and  diabetic  coma,  low  in  the  anaemias  of 
nephritis. 

Coagulation  Rate  of  Blood. — This  determination  is  of  value  in  con- 
nection with  operations  on  jaundiced  patients. 

Wright's  coagulometer  is  a  standard  instrument  but  is  cumbersome. 

A  simple  method  of  determining  the  rate  is  to  take  a-  piece  of  capil- 
lary glass  tubing  and  hold  it  downward  from  the  puncture  to  let  it 
fill  for  3  or  4  inches.  Then  at  intervals  of  thirty  seconds  scratch 
with  a  file  the  capillary  tubing  at  short  distances  and  break  off  between 
the  fingers.  When  coagulation  has  taken  place  a  long  worm-like  co- 
agulum  is  obtained.  Normally  coagulation  occurs  in  about  three  to 
four  ^minutes,  when  the  temperature  is  that  of  the  hand  in  which  the 
tubes  are  conveniently  held.  Rudolf  recommends  placing  the  tubes  in 
metal  tube  containers  in  a  Thermos  bottle  at  20°  C.  He  gives  the  normal 
coagulation  rate  for  this  temperature  as  8  minutes,  while  at  a  tempera- 
ture below  this  the  period  is  lengthened.  Age  and  sex  do  not  influence 
the  rate.  Sabrazes,  the  originator  of  this  method  found  no  appreciable 
variation  in  tubes  from  0.8  to  1.2  mm.  diameter. 


1 86  MICROMETRY   AND   BLOOD    PREPARATIONS 

In  Biirker's  test  you  mix  a  drop  of  blood  and  a  drop  of  distilled  water  on  a  slide 
and  with  a  capillary  tube  sealed  off  at  the  end  stir  the  mixture  every  half  minute. 
So  soon  as  fibrin  threads  appear  you  have  coagulation. 

SPECIFIC  GRAVITY  or  THE  BLOOD. 

Hammerschlag  has  a  method  for  the  determination  of  the  Hb. 
percentage  based  upon  the  specific  gravity  of  the  blood. 

In  this  method  a  mixture  of  benzol  and  chloroform  is  made  of  a  specific  gravity 
of  about  1050.  A  medium  size  drop  of  blood  is  then  taken  up  with  a  pipette  and 
dropped  into  the  mixture.  If  it  sinks  add  more  chloroform  from  a  dropping  bottle, 
if  it  tends  to  rise,  more  benzol.  The  mixture  in  which  the  drop  of  blood  tends  to 
remain  stationary,  near  the  top  of  the  mixed  benzol  and  chloroform,  has  the  same 
specific  gravity  as  that  of  the  blood.  This  is  determined  by  an  accurately  graduated 
hydrometer.  The  normal  average  specific  gravity  for  men  is  1059,  for  women  1056. 
A  table,  giving  the  Hb.  percentage  corresponding  to  the  specific  gravity  accom- 
panies the  outfit. 

To  determine  the  necessity  for  intravenous  infusion  in  cholera  Rogers  has  re- 
cently recommended  the  employment  of  small  bottles  containing  aqueous  solution 
of  glycerine  with  specific  gravities  varying  from  1048  to  1070,  increasing  the  specific 
gravity  in  each  successive  bottle  by  2°. 

An  accurate  urinometer  will  suffice  to  determine  the  specific  gravity.  Drops 
of  blood  from  the  cholera  patient  are  deposited  at  the  center  of  the  surface  of  the 
fluid  in  the  bottles  from  a  capillary  pipette.  If  the  specific  gravity  of  the  blood  is 
1062  at  least  a  liter  of  saline  or  sodium  bicarbonate  solution  is  needed.  If  1066  at 
least  two  liters.  Formerly  he  estimated  the  indications  by  blood  pressure  con- 
sidering a  pressure  of  80  in  Europeans  01  of  70  in  natives  as  indicating  intravenous 
injections. 

OCCULT  BLOOD. 

When  the  presence  of  blood  cannot  be  recognized  by  macroscopical 
or  microscopical  methods  (occult  blood)  we  must  resort  to  spectro- 
scopic  or  chemical  tests.  It  is  in  connection  with  blood  in  the  faeces 
that  these  tests  for  occult  blood  are  chiefly  called  for.  Before  making 
such  tests  on  faeces  it  is  advisable  to  have  the  patient  on  a  meat-free 
and  green-vegetable-free  diet  for  two  or  three  days.  It  is  chiefly  in 
carcinoma  or  ulcerations  of  the  gastro-intestinal  tract  that  such  exami- 
nations of  the  faeces  are  required. 

Haemin  Crystal  Test  (Teichman).— Prepare  a  solution  of  o.i  gram  each  of  KI, 
KBr,  and  KCL  in  100  c.c.  of  acetic  acid.  This  is  a  stable  solution.  Mix  some  of 
the  material  with  a  few  drops  of  the  solution  on  a  slide,  apply  a  cover-glass  and 
warm  the  material  until  bubbles  begin  to  appear  (gentle  steaming),  then  examine 
for  dark-brown  crystals. 


OCCULT  BLOOD  187 

Blood  in  the  Urine. — The  most  rapid  method  of  detection  is  by  using  the  micro- 
spectroscope.  An  ordinary  hand  spectroscope  will  answer  however. 

Donogany's  test  is  very  satisfactory.  To  10  c.c.  of  urine  add  i  c.c.  ammonium 
sulphide  solution  and  i  c.c.  of  pyridin.  The  urine  will  assume  a  more  or  less  deep 
orange  color  according  to  its  blood  content.  The  spectrum  of  alkaline  methaemo- 
globin  or  hgemochromogen  will  be  obtained.  See  illustrations  under  urine. 

In  making  the  guaiac  or  other  tests  it  is  a  good  plan  to  repeatedly  filter  the  blood- 
containing  urine  through  the  filter.  Then  touch  a  spot  on  the  moist  filter  with  the 
guaiac  or  benzidin  solution  and  then  finally  drop  on  this  so  treated  spot  a  drop  or 
two  of  hydrogen  peroxide  solution. 

Blood  in  Faeces  or  Gastric  Contents. — Take  5  grams  of  faeces  and  rub  it  up 
thoroughly  in  a  mortar  with  15  c.c.  of  a  mixture  of  equal  parts  of  alcohol,  glacial 
acetic  acid  and  ether.  Filter  through  an  unmoistened  pleated  filter  paper  re- 
peatedly until  only  3  to  4  c.c.  remain  of  the  filtrate.  The  faeces  filtrate  can  be 
first  tested  chemically  by  depositing  a  few  drops  in  the  center  of  3  or  4  circles  of 
white  filter-paper  placed  in  a  Petri  dish  or  upon  an  ordinary  white  plate. 

The  moistened  spot  is  then  treated  with  a  few  drops  of  a  freshly  prepared  alco- 
holic solution  of  guaiac  resin  (about  \  gram  of  guaiac  resin  is  broken  up  into 
small  fragments  and  shaken  up  in  about  3  c.c.  of  alcohol)  and  finally  there  is  drop- 
ped upon  the  spot  a  few  drops  of  a  solution  of  hydrogen  peroxide.  Waves  of  blue 
color  extending  out  into  the  moistened  filter-paper  show  a  positive  test  for  blood. 

For  the  benzidin  test  pour  on  this  fasces  filtrate-moistened  filter-paper  a  few 
drops  of  the  following  solution:  2  c.c.  of  a  saturated  alcoholic  solution  of  benzidin 
2  c.c.  of  solution  of  peroxide  of  hydrogen  and  two  drops  of  glacial  acetic  acid.  (Blue.) 

If  the  aloin  test  is  preferred  we  treat  the  filtrate-moistened  filter-paper  with  a 
few  drops  of  a  3%  solution  of  aloin  in  70%  alcohol  and  then  treating  the  spot 
with  hydrogen  peroxide  solution.  Brick  red  colour. 

More  reliable  is  the  spectroscopic  test.  For  this  we  take  about  3  c.c. 
of  the  concentrated  ether,  acetic  acid,  alcohol  faecal  nitrate  and  add  to  it 
2  c.c.  of  pyridin.  Then  add  not  more  than  2  to  3  drops  of  ammonium 
sulphide  solution.  (The  ammonium  sulphide  solution  should  be  kept 
in  an  amber-coloied,  glass-stoppered  bottle.  The  solution  should  be 
freshly  prepared  every  10  days.)  Examine  the  solution,  contained  in  a 
small  test-tube,  with  the  spectroscope  and  the  two  absorption  bands  of 
methaemoglobin-alkaline  (haemochromogen),  between  D  and  E,  show 
a  positive  blood  test.  Comparison  should  be  made  with  fresh  blood, 
in  which  the  absorption  band  in  the  yellow  is  nearer  line  D 
(oxyhaemoglobin  spectrum). 


CHAPTER  XIV. 
NORMAL  AND  PATHOLOGICAL  BLOOD. 

IN  considering  what  may  be  termed  normal  blood,  it  must  be  borne 
in  mind  that  the  normal  varies  for  men,  women,  and  children: 

Hb.  Red  cells.  Leukocytes. 

Men,  90  to  110%,  5  to  5  1/2  million,  7500. 
Women,  80  to  100%,  4  1/2  to  5  million,  7500. 
Children,  70  to  80%,  4  1/2  to  5  million,  9000. 

COLOR  INDEX. 

This  is  obtained  by  dividing  the  percentage  of  the  haemoglobin  by 
the  percentage  of  red  cells,  five  million  red  cells  being  considered  as 
100%.  To  obtain  the  percentage  of  red  cells  it  is  only  necessary  to 
multiply  the  two  extreme  figures  to  the  left  by  two.  Thus  if  a  count 
showed  the  presence  of  1,700,000  red  cells,  the  percentage  would  be 
34  (17X2=34).  If  the  Hb.  percentage  in  this  case  were  50;  then  the 
color  index  would  be  50-^34,  or  1.4. 

In  normal  blood  the  color  index  is,  approximately,  i. 

In  anaemias  we  have  three  types  of  color  index:  i.  The  pernicious 
anaemia  type,  which  is  above  i.  Here  we  have  a  greater  reduction  in 
red  cells  than  we  have  of  the  haemoglobin  content  of  each  cell.  2.  The 
normal  type,  when  both  red  cells  and  haemoglobin  are  proportionally 
decreased,  as  in  anaemia  following  haemorrhage.  3.  The  chlorotic  type. 
Here  there  is  a  great  decrease  in  haemoglobin  percentage,  but  only  a 
moderate  decrease  in  the  number  of  red  cells.  Hence  the  color  index 
is  only  a  fraction  of  i.  For  example,  in  a  case  of  chlorosis  we  have  40% 
of  haemoglobin  and  90%  of  red  cells,  40^90  =  0.4. 

RED  CELLS. 

In  considering  the  corpuscular  richness  of  a  specimen  of  blood,  it 
must  be  remembered  that  this  does  not  necessarily  bear  any  relation  to 
the  quantity  of  blood  in  the  body.  Thus,  a  more  or  less  bloodless- 


PATHOLOGICAL   RED    CELLS  189 

looking  individual,  the  total  quantity  of  whose  blood  is  greatly  reduced, 
may,  notwithstanding,  give  a  normal  red  count.  In  examining  a  speci- 
men of  peripheral  blood  we  get  a  qualitative,  not  a  quantitative  result. 

Normally,  we  have  an  increase  in  red  cells  in  those  living  at  high  altitudes. 
An  altitude  of  two  thousand  feet  may  increase  the  red  count  about  one  million,  and 
a  height  of  six  thousand  feet  about  two  million.  Profuse  sweats  and  diarrhoeas  also 
increase  the  red  count.  Pathologically,  in  chronic  polycythemia  with  cyanosis 
and  splenic  enlargement,  we  have  a  red  count  of  about  ten  million.  In  cyanosis 
from  heart  disease,  etc.,  and  in  Addison's  disease  there  is  also  an  increase  in  red 
cells. 

The  normal  red  cell  or  ery throcyte  measures  about  7.5^  in  diameter.  It  is  non- 
nucleated  and  normally  stains  with  acid  dyes,  taking  the  pink  of  eosin  or  the  orange 
of  orange  G.  If  larger,  10  to  20;*,  it  is  called  a  macrocyte;  if  smaller,  3  to  6/*,  a 
microcyte. 

Anisocytosis  is  a  term  applied  to  a  condition  where  marked  varia- 
tion in  size  of  the  red  cells  occurs. 

Macrocytes  are  rather  indicative  of  severe  forms  of  anaemia,  the 
microcytes,  of  less  grave  types.  When  the  red  cell  is  distorted  in  shape, 
it  is  called  a  poikilocyte.  Care  must  be  exercised  that  distorted  shapes 
are  not  due  to  faulty  technic.  Crenation  and  vacuolation  of  red  cells 
are  marked  in  poorly  prepared  specimens. 

In  addition  to  variation  in  size  and  shape,  we  also  have  pathological 
variation  in  staining  affinities. 

Polychromatophilia.— This  shows  itself  by  red  cells  taking  a  brown- 
ish to  a  dirty  blue  tint,  as  is  frequently  seen  in  immature  red  cells, 
especially  nucleated  ones. 

Granular  basophilic  degeneration  (also  termed  punctate  baso- 
philia  and  stippling)  refers  to  the  presence  of  blue  dots  in  the  pink  back- 
ground of  stained  red  cells.  It  is  found  in  many  severe  anaemias,  as 
pernicious  anaemia,  the  leukaemias,  malarial  cachexia,  etc.  It  is  very 
characteristic  of  lead  poisoning. 

The  nucleated  red  cell,  while  normal  for  the  marrow,  is  always 
pathological  for  the  blood  of  the  peripheral  circulation.  Normoblasts 
have  the  diameter  of  a  normal  red  cell.  The  nucleus  is  round  and  stains 
intensely  with  basic  dyes,  often  appearing  almost  black.  Another 
characteristic  is  that  it  frequently  appears  as  does  the  setting  in  a  ring. 
Some  give  the  term  microblast  to  smaller  nucleated  forms.  In  normo- 
blasts  the  red  cell  proper  stains  normally.  The  megaloblasts  not  only 
have  a  greater  diameter  than  the  normoblast,  but  the  nucleus  is  poor 
in  chromatin,  stains  less  intensely  and  is  less  distinctly  outlined.  In- 


I QO  NORMAL    AND    PATHOLOGICAL  BLOOD 

stead  of  being  round,  the  nucleus  is  irregular  and  may  be  trefoil  in  shape. 
The  cytoplasm  surrounding  the  nucleus  shows  polychromatophilia. 
This  contrasted  with  the  pure  blue  of  the  lymphocytes  should  differen- 
tiate. Nornioblasts  are  found  in  secondary  anaemias,  and  especially  in 
myelogenous  leukaemia.  Megaloblasts  are  peculiarly  characteristic  of 
pernicious  anaemia.  Enormous  megaloblasts  are  sometimes  termed 
gigantoblasts. 

In  aplastic  anaemia  (a  severe  type  of  pernicious  anaemia),  in  contrast  to  ordinary 
pernicious  anaemia,  nucleated  reds  are  very  rarely  found.  There  is  also  very  little 
poikilocytosis,  and  the  color  index  is  about  normal.  It  is  a  rare,  rapidly  fatal 
anaemia,  particularly  of  young  women. 

It  does  not  show  remissions,  runs  a  rapid  course,  and  is  attended  with  a  marked 
increase  of  lymphocytes.  The  bone  marrow  of  the  femur  is  pinkish  yellow  and 
homogeneous. 

The  term  leukanaemia  has  been  employed  to  describe  conditions 
which  partake  of  the  characteristics  of  pernicious  anaemia  and  leukaemia. 

WHITE  CELLS. 

Owing  to  the  conflicting  views  as  to  origin,  nature,  and  functions  of 
the  various  leukocytes,  their  classification  is  in  a  state  of  confusion. 
As  regards  the  appearance  of  the  cells,  this  of  course  varies  as  the  stain 
used,  and  it  requires  considerable  experience  for  a  single  individual  to  be 
able  to  positively  recognize  the  difference  between  a  lymphocyte  and 
a  large  mononuclear  when  one  specimen  is  stained  with  a  Romanowsky 
stain,  another  with  Ehrlich's  triacid,  and  a  third  with  haematoxylin  and 
eosin.  This,  of  course,  is  intensified  when  different  persons  adhere  to 
the  method  of  staining  which  they  prefer  and  are  at  a  loss  to  appreciate 
differences  which  are  brought  out  by  some  other  stain  used  by  some 
other  person.  Even  with  the  same  stain  used  with  different  specimens 
of  blood  we  find  the  staining  characteristics  of  various  leukocytes  imper- 
ceptibly merging  the  one  into  the  other,  so  that  at  times  it  is  impossible 
for  one,  even  with  his  own  standard  of  differentiation,  to  be  sure  whether 
he  is  dealing  with  a  lymphocyte  or  a  laige  mononurlear.  The  difficulty 
is  even  greater  when  we  deal  with  Turck's  irritation  forms  and  with 
myelocytes. 

Without  going  into  the  various  granule  stainings  so  thoroughly 
brough  out  by  Ehrlich,  we  shall  immediately  take  up  the  question  of 
a  practical  classification  for  use  in  making  a  differential  count.  As  the 
Romanowsky  method  of  staining  (Wright,  Leishman,  or  Giemsa)  gives 


THE    LEUCOCYTES  IQI 

us  information  not  yielded  by  either  haematoxylin  and  eosin  or  the  tri- 
acid,  the  points  of  differentiation  to  be  referred  to  in  that  which  follows 
is  with  blood  so  stained. 

In  considering  the  staining  affinities  of  different  parts  of  the  leuko- 
cytes1, it  is  convenient  to  divide  such  into  basic  ones,  acid  ones,  and  thos-e 
which  may  be  said  to  be  on  the  border  line  between  these — the  so-called 
neutrophilic  affinities. 

With  Wright's  stain  we  have  the  eosinophile  or  oxyphile  affinity  of 
the  granules  of  eDsinophiles  for  acid  dyes,  in  this  case  eosin.  The 
nuclei  and  basophile  granules  have  affinities  in  greater  or  less  degree  for 
basic  stains  (the  blue  and  the  violet  shading  resulting  from  methylene 
blue  as  modified  by  poly  chroming) .  With  the  granules  in  the  cyto- 
plasm of  the  poly morphonucl  ears  and  neutrophilic  myelocytes,  and  to  a 
less  extent  in  the  transitional,  we  have  a  staining  which  merges  into  a 
yellowish-red  on  the  one  extreme  and  into  a  lilac  on  the  other.  As  a 
standard,  neutrophilic  granules  should  be  a  mean  of  these  extremes. 

Not  only  by  reason  of  the  authority  of  Ehrlich,  but  because  such  a 
division  gives  all  variations,  which  can  then  be  combined  by  one  pre- 
ferring a  simpler  classification,  it  would  seem  proper  to  divide  the  nor- 
mal leukocytes  into : 

1.  Small  Lymphocytes. — These  are  small  round  cells  about  the  size 
of  a  red  corpuscle  with  a  large  centrally  placed,  deeply  violet  staining 
nucleus  and  a  narrow  zone  of  cytoplasm.     This  cytoplasm  may  not  be 
more  than  a  mere  crescentic  fringe.     This  is  the  type  of  lymphocyte 
which  makes  up  the  greater  proportion  of  the  leukocytes  in  chronic 
lymphatic  leukaemia.     At  times  these  cells  seem  to  be  composed  of 
nucleus  alone. 

2.  Large  Lymphocytes. — These  are  of  the  same  type  as  small 
lymphocytes,   but  possessing  more  cytoplasm.     The  nucleus,   while 
round  and  taking  a  fairly  deep  rich  violet  stain,  does  not  stain  so  deeply 
as  the  nucleus  of  the  small  lymphocytes.     The  cytoplasm  is  a  clear, 
translucent,  pure  blue.     It  may  contain  pinkish  granules  known  as  azur 
granules,  but  these  are  of  rathei  large  size  and  do  not  mar  the  glass-like 
appearance.     They  are  from  9  to  15^  in  diameter  and  are  common  in 
children.     In  the  acute  lymphatic  leukaemias  they  at  times  predominate. 

3.  Large  Mononuclears. — -These  are  large  round  or  oval  cells  with 
a  nucleus  which  has  lost  the  richness  of  violet  staining  of  the  lymphocyte 
nucleus.     The  nucleus  is  furthermore  frequently  irregular  in  outline 
or  may  show  the  commencing  indentation  of  the  transitional  nucleus. 


1 92  NORMAL   AND    PATHOLOGICAL  BLOOD 

There  is  not  that  sharp  distinction  between  nucleus  and  cytoplasm 
that  exists  in  the  lymphocytes.  The  cytoplasm  of  the  large  mononu- 
clear  gives  the  impression  of  opacity,  as  if  it  were  frosted  glass  instead 
of  clear  glass.  The  neutrophile  mottling  which  begins  to  appear  causes 
a  disappearance  of  the  pure  blue  character  of  the  cytoplasm  of  the 
lymphocyte.  It  is  principally  by  the  washed-out  staining  of  the  nucleus 
and  the  opaque  lilac  of  the  cytoplasm  that  we  differentiate  them  from 
the  lymphocytes.  They  greatly  resemble  Tiirck's  irritation  forms  or 
plasma  cells  and  may  be  confused  with  myelocytes. 

4.  Transitionals. — These  appear  as  but  a  later  stage  in  the  decay 
of  the  large  mononuclears;  the  nucleus  is  more  indented,  frequently 
horseshoe-shaped,  and  has  a  washed-out  violet  shade  of  less  intensity 
than  that  of  the  large  mononuclears.  These  are  the  cells  so  often  dis- 
rupted in  smears. 

These  four  kinds  of  cells  are  frequently  referred  to  as  the  lymphocyte 
series,  and  although  many  authorities  consider  that  the  small  lympho- 
cyte represents  a  more  mature  cell  than  the  others  of  this  class,  yet  it 
is  thought  by  others  that  the  age  of  the  cell  increases  as  we  go  from  small 
lymphocytes  to  large  lymphocytes,  thence  to  the  large  mononu clear; 
and  then  in  the  transitional  we  have  the  decrepit  stage  which  precedes 
dissolution.  The  old  view  that  the  transitional  was  the  precursor  of 
the  polymorphonuclear  has  few  advocates  at  the  present  time. 

While  it  is  convenient  to  consider  these  hyaline  cells  as  representing 
different  stages  in  development,  yet  from  a  standpoint  of  immunity  this 
is  untenable.  The  large  mononuclears  and  transitionals  are  the  cells 
in  which  we  find  certain  animal  cells  and  pigment  phagocytized,  as  is 
the  case  in  malaria.  These  cells  are  the  macrophages  of  Metchnikoff 
and  are  probably  derived  from  the  bone  marrow. 

The  lymphocytes  take  origin  from  the  lymphoid  tissue,  and  very 
probably  the  large  lymphocyte  is  a  younger,  more  immature  cell  than 
the  small  lymphocyte. 

Ehrlich  and  Naegeli  regard  the  large  mononuclears  as  of  myeloid 
origin  while  Pappenheim  considers  them  to  belong  to  the  group  of 
lymphocytes. 

A  normal  percentage  of  large  mononuclears  and  transitionals  com- 
bined should  not  exceed  about  4%. 

In  addition  to  the  series  of  leukocytes  just  considered  we  have  pres- 
ent normally  in  the  blood  three  types  of  granular  cells  distinguished 
according  to  the  staining  affinity  of  their  granules.  These  are: 


ARNETH  INDEX  1 93 

1.  Polymorphonuclear  Leukocytes. — -This  cell  normally  constitutes 
the  greater  proportion  of  the  leukocytes.     It  is  an  amoeboid,  actively 
phagocytic  cell,  about  10  or  12  /*  in  diameter,  and  is  the  microphage  of 
Metchnikoff.     Bacteria  are  actively  phagocytized  by  this  cell,  and  it  is 
the  cell  concerned  in  determining  the  opsonic  power  of  blood  to  various 
bacteria.     It  has  fine  lilac  granules  which  are  termed  neutrophilic  (ep- 
silon  granules).     The  single  nucleus  is  rich  in  chroma  tin  and  is  lobose 
like  the  kernel  of  an  English  walnut;  frequently  it  resembles  the  letter 
z.     These  cells  are  derived  from  the  neutrophilic  myelocytes  of  the  bone 
marrow.     It  is  in  these  cells  that  the  glycogen,  or  iodophil  granules, 
appear  in  certain  suppurative  conditions. 

A  great  deal  of  interest  has  been  aroused  in  the  so-called  Arneth  index,  espe- 
cially in  connection  with  prognosis  in  tuberculosis  and  various  pyogenic  infections. 
The  basis  of  the  test  is  that  polymorphonuclears  showing  only  one  or  two  nuclear 
nodes  are  considered  immature  while  those  having  three,  four  or  five  nuclear  nodes 
possess  greater  phagocytic  power. 

A  normal  distribution  is  as  follows: 

Class  I.  Class  II.  Class  III.  Class  IV.  Class  V. 

6%  35%  42%  16%  i% 

To  obtain  the  Arneth  index  add  to  the  sum  of  the  polymorphonuclear  percent- 
ages of  cells  containing  one  and  two  nodes  one-half  of  the  percentage  of  those  having 
three  nodes.  In  the  above  we  have  as  the  normal  Arneth  index  62. 

In  an  advanced  case  of  tuberculosis  we  might  have  an  index  of  79,  obtained  as 
follows : 

Class  I.  Class  II.  Class  III.  Class  IV.  Class  V. 

20%  45%  28%  6%  i% 

2.  Eosinophile  Leukocytes.— These  are  very  striking  cells  with 
coarse  granules  staining  brilliantly  pink,  the  eosinophile,  oxyphile,  or 
acidophile  granules  (alpha  granules  of  Ehrlich).     The  cells  are  a  little 
larger  than  the  polymorphonuclears.     The  normal  eosinophile  is  to  be 
distinguished  from  the  eosinophilic  myelocyte  by  its  possessing  two 
distinct  lobes  in  the  nucleus.     At  times  we  find  three  nuclei.     The 
nucleus  of  the  myelocyte  is  round.     The  eosinophile  is   the  cell  so 
frequently  increased  in  infections  by  intestinal  animal  parasites. 

3.  Mast  Cells.— These  also  have  coarse  granules,  but  they  stain  a 
deep  violet  blue.     Hence  they  are  basophile  granules  (gamma  granules). 
In  fresh  blood  these  granules  do  not  show  up  very  well,  thus  they  can  be 
distinguished  from  the  highly  refractile  granules  of  the  eosinophile. 

13 


194  NORMAL  AND   PATHOLOGICAL  BLOOD 

The  trilobed  nucleus  stains  less  intensely  than  the  granules.     As  a  rule, 
the  mast  cell  is  about  the  size  of  a  polymorphonuclear. 

In  a  differential  count  of  normal  blood  we  find  about  the  following  percentages. 

Polymorphonuclears,  65  to  70%,  about  5000  per  c.  mm. 

Small  lymphocytes,  20  to  25%,  about  1500  per  c.  mm. 

Large  lymphocytes,  5  to  10%,  about  500  per  c.  mm. 

Large  mononuclears,  i  to    2%,  about  100  per  c.  mm. 

Transitionals,  2  to    4%,  about  200  per  c.  mm. 

Eosinophiles,  i  to    2%,  about  TOO  per  c.  mm. 

Mast  cells,  1/4  to  1/2%,  about  25  per  c.  mm. 

The  leukocytes  which  are  found  in  the  peripheral  circulation  only 
in  pathological  additions  are: 

1.  Neutrophilic  Myelocytes.— The  common  type  is  a  large  cell 
with  a  large  centrally  placed,  feebly  staining  nucleus.     This  may  be 
recognized  by  the  difficulty  of  distinguishing  the  nucleus  from  the  cyto- 
plasm, there  being  no  sharp  line  separating  these  parts  of  the  cell.     They 
imperceptibly  merge  into  one  another.     They  differ  from  a  large  mono- 
nuclear  in  that  the  cytoplasm  is  distinctly  dotted  with  neutrophile 
granules  and  that  we  cannot  make  out  a  distinct  line  of  separation  of  a 
slightly  irregular  or  indented  nucleus  from  the  surrounding  slightly 
neutrophilic  cytoplasm.     Cornil  has  described  a  very  large  myelo.cyte 
with  eccentrically  placed  nucleus  and  neutrophilic  granules. 

Myelocytes  are  at  times  found  with  both  basophilic  and  neutrophilic 
granules,  and  may  rarely  be  seen  to  have  all  three  kinds  of  granules  on  a 
single  myelocyte,  acidophile,  basophile,  and  neutrophile. 

2.  Eosinophilic   Myelocytes. — These    can   be   distinguished   from 
normal  eosinophiles  by  their  possessing  a  single  round  nucleus,  not 
bilobed.     These  myelocytes  may  be  as  large  as  a  normal  eosinophile, 
but  frequently  are  no  larger  than  a  red  cell. 

The  neutrophile  myelocyte  is  characteristic  of  spleno-myelogenous 
leukaemia,  the  eosinophile  one  ofmyelogenic  leukaemia.  The  occurrence 
of  an  occasional  myelocyte  is  frequently  noted  in  conditions  having  a 
leukocytosis.  In  diphtheria  their  presence  in  numbers  is  of  bad  prog- 
nostic import.  Myelocytes  are  of  diagnostic  importance  in  metastases 
of  malignant  tumors. 

3.  The  Irritation  Cell  of  Turck,  or  Plasma  Cell,— This  cell  has  a 
faintly  staining,  eccentrically  placed  nucleus,  and  a  dark  opaque  blue, 
frequently  vacuolated,  cytoplasm.     They  are  usually  recorded  as  large 


BLOOD   PLATELETS  I 95 

mononuclears.     Turck  supposed  them  to  appear  in  the  circulation  as 
the  result  of  bone-marrow  irritation. 

4.  Myeloblasts. — These  cells  are  found  in  myeloid  leukaemia  and 
though  often  mistaken  for  lymphocytes  or  large  mononuclears  they  are  of 
marrow  origin.     The  nucleus  stains  more  intensely  than  that  of  the 
large  mononuclear  and  the  cytoplasm  is  more  deeply  blue  stained  than 
that  of  the  large  lymphocyte.     They  also  contain  three  or  four  nucleoli. 

Pyronin  methyl-green  staining  is  best  for  demonstrating  the  nuclei. 

5 .  Pathological  Large  Lymphocytes. — These  are  as  a  rule  much  larger 
than  normal  large  lymphocytes  and  show  poorer  staining  of  both 
nucleus   and    cytoplasm.     The  nuclei  often  show  the  appearance  of 
division  into  two  or  more  lobes,  thus  showing  the  characteristics  of 
Rieder  cells.     They  may  be   confused  with  large  mononuclears  but 
are  considered  to  be  derived  from  the  germinal  centers  of  various  lym- 
phoid  tissues.     They  are  found   in   leukaemic   and   pseudo-leukaemic 
conditions. 


BLOOD  PLATELETS. 

These  are  normally  present  in  blood  in  the  number  of  about  350,000  per  cubic 
millimeter.  They  disintegrate  very  quickly  after  the  blood  is  withdrawn.  Wright 
has  demonstrated  that  they  are  pinched-off  projections  of  giant  cells  of  the  bone 
marrow.  They  consist  only  of  protoplasm,  no  nuclear  material.  They  do  not 
contain  haemoglobin.  In  conditions  where  giant  cells  are  less  abundant,  as  in  per- 
nicious anaemia,  the  blood  platelets  are  less  abundant.  In  myelogenous  leukaemia 
they  are  very  abundant.  They  vary  in  size  from  2  to  5/f  according  as  a  larger  or 
smaller  pseudopod  of  a  giant  cell  has  been  broken  off.  Stained  with  Wright's  stain, 
they  are  more  purplish  than  blue  and  show  thread-like  projections.  They  are  often 
mistaken  for  the  protozoal  causes  of  various  diseases.  Especially  are  they  confused 
with  malarial  parasites  when  lying  on  a  red  cell.  The  blood  plate  has  no  brick- 
red  chromatic  material;  it  is  purplish  rather  than  blue,  and  has  no  pigment  grains. 
It  is  advisable  to  compare  these  isolated  blood  plates  with  the  larger  or  smaller  ag- 
gregations scattered  about  the  smears.  In  this  way  their  true  character  is  apparent. 
In  addition  to  blood  platelets,  which  in  fres.h  blood  can  only  be  observed  when  a 
fixative  is  used,  we  have  other  confusing  bodies. 

The  haemokonia  of  Muller  are  small,  highly  refractile  bodies  showing  active 
oscillatory  movement.  They  are  supposed  to  be  cast-off  granules  of  eosinophiles 
or  other  leukocytes,  or  possibly  derived  from  nuclei.  As  this  blood  dust  or  haemo- 
konia is  found  in  a  marked  degree  in  lipaemia  it  may  be  that  the  particles  are  fat. 
It  is  interesting  that  this  lipaemia  is  absent  after  the  taking  of  large  quantities  of 
fat  in  cases  with  serious  pancreatic  trouble.  The  serum  of  a  normal  individual  is 
rather  turbid  after  slight  indulgence  in  butter.  Pinched-off  fragments  of  red  cells 
may  also  appear  as  possible  protozoal  bodies. 


196  NORMAL  AND  PATHOLOGICAL  BLOOD 

LEUKOPENIA. 

This  is  a  term  used  to  designate  a  reduction  in  the  normal  number 
of  leukocytes.  A  leukocyte  count  of  5000  would  represent  a  slight 
leukopenia;  one  of  2000,  a  marked  leukopenia.  In  the  later  stages 
of  typhoid,  and  in  acute  miliary  tuberculosis,  we  expect  a  moderate 
leukopenia. 

The  leukopenia  of  typhoid  is  moderate  and  is  often  preceded  in  the  first  few 
days  by  a  moderate  neutrophile  leukocytosis.  Later  on  we  have  a  decided  increase 
in  the  lymphocytes.  A  marked  diminution  or  absence  of  eosinophiles  is  so  character- 
istic that  any  increase  in  eosinophilic  percentage  negatives  a  diagnosis  of  typhoid. 

Paratyphoid  gives  a  similar  blood  picture. 

Chronic  alcoholism  and  chronic  arsenic  poisoning  cause  a  reduction 
in  the  number  of  the  white  cells.  Pernicious  anaemia  shows  a  marked 
leukopenia,  as  is  also  the  case  with  Band's  disease.  Two  tropical  dis- 
eases, kala-azar  and  dengue,  show  a  marked  leukopenia,  the  counts 
often  being  below  2500.  During  the  apyrexial  period  of  malaria  we 
may  have  a  white  count  of  5000,  ^ 

It  has  recently  been  claimed  that  a  leukopenia  with  a.  coincident 
marked  reduction  in  the  lymphocytes  is  characteristic  of  ifceasles  and 
that  this  occurs  several  days  before  the  Koplik  spots  appear?*v 


Kocher  notes  that  in  exophthalmic  goiter  the  leukocyte  count  is  considerably 
diminished  and  that  the  polymorphonuclears  are  not  much  more  than  one-half  the 
usual  percentage  while  the  percentage  of  the  lymphocytes  is  almost  double  the 
normal. 

X-ray  treatment  tends  to  destroy  leukocytes  in  the  exposed  region,  especially 
polymorphonuclears.  The  small  lymphocytes  are  least  affected. 

EOSINOPHILIA. 

Where  the  eosinophiles  are  increased  to  5%,  we  have  a  moderate 
eosinophilia.  In  some  cases  of  infection  with  intestinal  parasites, 
especially  hook-worms,  but  also  from  other  parasites,  as  round  and 
whip-worms,  we  may  have  an  eosinophilia  of  30  to  50%.  In  Guam, 
among  the  natives,  it  is  difficult  to  find  an  eosinophile  count  under  15%. 
The  eosinophilia  tends  to  disappear  when  the  anaemia  becomes  very 
severe. 

The  eosinophilia  of  trichinosis  is  best  known,  and  a  combination  of 
this  blood  finding  with  fever  and  marked  pains  of  mucles,  would  justify 
the  excision  of  a  piece  of  muscle  for  examination  for  encysted  embryos. 


LEUCOCYTOSIS  197 

In  true  asthma  eosinophilia  is  marked,  and  its  absence  is  of  value  in  indi- 
cating other  causes  for  the  condition.  Certain  skin  diseases,  especially 
pemphigus,  show  eosinophilia. 

Eczema  and  psoriasis  are  not  apt  to  give  more  than  3  or  4%  eosinophiles.     A 
rather  high  degree  of  eosinophilia  is  found  in  mycosis  fungoides. 
Scabies  also  gives  an  eosinophilia. 

The  proportion  of  eosinophiles  in  the  blood  of  children  is  greater 
than  in  that  of  adults. 

Increase  of  both  eosinophiles  and  mast  cells  is  found  in  myelogenous 
leukaemia. 

LEUKOCYTOSIS. 

It  is  to  an  increase  in  the  polymorphonuclears  that  this  term  is 
usually  applied,  the 'term  lymphocytosis  or  eosinophilia  being  employed 
where  ^vvhite  cells  of  eosinophile  or  lymphocyte  nature  are  increased. 
We  have  physiological  leukocytosis  in  the  latter  weeks  of  pregnancy, 
also  in  the  new-born,  and  in  connection  with  digestion. 

Pathological  Leukocytosis.—  Pneumonia.  In  this  disease  we  have  a 
leukocytosis  of  20,000  to  30,000  or  higher.  The  eosinophiles  are  almost 
absent.  A  normal  leukocyte  count  in  pneumonia  makes  a  prognosis 
unfavorable. 

The  leukocyte  count  drops  about  the  time  of  the  crisis,  and  with 
the  reappearance  of  eosinophiles  is  a  favorable  sign.  A  moderate 
leukocytosis  occurs  in  carcinoma  and  sarcoma. 

Septic  processes.  The  leukocyte  count  is  of  great  value,  especially 
when  we  obtain  a  leukocytosis  with  80  to  90%  of  polymorphonuclears, 
as  in  appendicitis,  cholecystitis,  or  other  suppurative  conditions. 

According  to  Cabot,  leukocytosis  varies  in  infections  as  follows: 

1.  Severe  infection — good  resistance;  early,  marked  and  persistent  leuko- 
cytosis. 

2.  Slight    infection — slight    resistance;    leukocytosis    present,    but    not 
marked. 

3.  In  fulminating  infections  we  may  have  no  increase  in  whites,  but  a 
higher  percentage  of  polymorphonuclears. 

4.  Slight  infection  and  good  resistance  may  not  be  productive  of  leuko- 
cytosis. 

It  is  in  connection  with  the  question  of  operation  in  appendicitis 
or  similar  conditions  that  the  matter  of  a  leukocyte  count  is  of  prime 
importance.  If  there  be  a  leukocytosis  but  with  less  than  75%  of 


198  NORMAL   AND    PATHOLOGICAL  BLOOD 

polymorphonuclears  it  indicates  an  infection  of  little  virulence  or  a 
walled-off  process  with  an  exacerbation.  It  is  difficult  to  form  an  opin- 
ion when  the  polymorphonuclears  are  under  80%.  Leukocytosis  with 
polymorphonuclear  percentage  of  85  to  90  indicates  immediate  opera- 
tion; percentages  over  90  point  to  peritonitis  and  if  with  such  per- 
centages of  polymorphonuclears  there  is  absence  of  leukocytosis  the 
prognosis  is  grave. 

Spirochaeta  fevers,  as  relapsing  fever,  may  give  a  leukocytosis  of 
from  25,000  to  50,000. 

Smallpox,  especially  at  time  of  pustulation,  plague,  scarlet  fever, 
and  liver  abscess  give  a  leukocytosis  of  from  12,000  to  15,000. 

Smallpox    often    shows  a  very  large  percentage  of  very  characteristic  large 
mononuclears. 


FIG.  54. — Leukocytosis  (40,000);  sixteen  polymorphonuclears  in  field.     (Cabot.} 

The  leukopenia  and  lymphocyte  increase  in  measles  are  important  points  in 
differentiating  it  from  scarlatina. 

With  meningitis  counts  of  25,000  are  not  unusual,  in  abscess  of  the 
brain  the  white  count  rarely  exceeds  15,000. 

Poliomyelitis  and  polioencephalitis  give  a  slight  leukocytosis  during 
the  febrile  accession. 

Erysipelas  and  epidemic  cerebrospinal  meningitis  also  give  a  leukocytosis  of 
from  15,000  to  20,000.  In  malignant  diseases  we  sometimes  have  a  moderate 
leukocytosis.  Rogers  states  that  in  liver  abscess,  with  a  leukocytosis  of  15,000  to 
20,000,  we  have  only  about  75  to  77%  of  polymorphonuclears — there  being  also 
a  moderate  increase  in  the  percentage  of  large  mononuclears. 

Drugs  such  as  antipyrin  may  give  a  leukocytosis.  The  leukocyte  increase  of 
pilocarpine  is  rather  a  lymphocytosis. 


THE  PRIMARY  ANAEMIAS  199 

LYMPHOCYTOSIS. 

Of  course,  the  disease  in  which  we  have  the  most  marked  lymphocy- 
tosis  is  lymphatic  leukaemia. 

The  lymphocytosis  of  typhoid  fever  has  been  taken  up  under  leuko- 
penia. 

Whooping-cough  may  give  a  lymphocytosis  of  20,000  to  30,000. 

Young  children  have  normally  an  excessive  proportion  of  lymphocytes.  This 
is  apt  to  be  particularly  marked  in  hereditary  syphilis.  Enlarged  tonsils  may 
give  rise  to  a  lymphocytosis  of  10,000  to  15,000,  when  more  than  50%  of  the  white 
cells  will  be  lymphocytes.  Rickets  and  scurvy  give  a  lymphocytosis. 

Varicella  and  mumps  may  also  give  a  a  increase  in  the  percentage  of 
lymphocytes. 

Malta  fever  is  a  disease  which  may  show  quite  a  mononuclear  increase. 

DISEASES  IN  WHICH  THERE  is  A  NORMAL  LEUKOCYTE  COUNT. 

Uncomplicated  tuberculosis,  influenza,  Malta  fever,  measles,  try- 
panosomiasis,  malaria,  syphilis,  and  chlorosis.  In  malaria  we  have 
a  leukocytosis  at  the  time  of  the  rigor,  while  during  the  apyrexial  period 
there  is  a  moderate  leukopenia.  In  malaria  we  have  a  marked  increase 
in  the  percentage  of  the  large  mononuclears  and  transitionals.  These 
may  form  from  25 %  to  35 %  of  the  leukocytes.  When  beaiing  particles 
of  pigment  they  are  known  as  melaniferous  leukocytes — macrophages 
which  have  ingested  malarial  material.  In  dengue,  at  the  time  of  the 
terminal  rash,  we  may  have  as  great  a  percentage  of  large  mononuclears. 
In  this  disease,  however,  we  have  a  great  diminution  of  polymorphonu- 
clears  from  the  start  (25  to  40%).  Instead  of  a  large  mononuclear  we 
have  at  the  onset  a  lymphocytic  increase.  There  is  an  increase  of  large 
mononuclears  in  trypanosomiasis. 

The  white  count  is  about  normal  in  uncinariasis  (Ashford's  average 
was  7800).  Some  have  reported  a  leukopenia  in  severe  cases. 

While  eosinophilia  is  the  most  marked  feature  in  hook-worm  disease 
yet  in  very  severe  cases  it  may  be  absent. 

THE  PRIMARY  ANAEMIAS. 

Chlorosis. — In  chlorosis  it  is  the  reduction  of  haemoglobin  with  the 
slight  numerical  variation  from  normal  of  the  red  cells  that  makes  for 
a  diagnosis.  The  color  index  is  very  low.  There  is  nothing  abnormal 


200  NORMAL   AND   PATHOLOGICAL  BLOOD 

about  the  leukocytes.  Microcytes  may  be  present,  and  very  occasion- 
ally a  normoblast.  Macrocytes  and  megaloblasts  are  always  absent. 
Blood  of  chlorotics  is  very  pale  and  very  fluid  and  coagulates  rapidly, 
hence  frequency  of  thrombosis. 

Spleen,  liver,  and  lymph  glands  as  a  rule  normal. 

Simple  Primary  Anaemia. — This  condition  is  not  recognized  by  many  authors, 
but  is  a  convenient  term  under  which  to  group  anaemias  which  are  neither  chlorosis 
nor  pernicious  anaemia  and  for  which  no  assignable  cause  can  be  designated.  It 
is  a  secondary  anaemia  without  a  cause.  In  it  color  index  is  about  normal,  there 
is  no  change  in  the  leukocytes  and  cases  go  on  to  recovery. 

Pernicious  Anaemia. — In  pernicious  anaemia  we  obtain  a  very 
fluid,  but  normally  colored  drop  of  blood  upon  puncture.  The  yellow 


FIG.  55. — Pernicious  anaemia.     M.m,   Megaloblasts;    n,    normoblast;   s,    stippling 
(punctate  basophilia).     (Cabot.) 

marrow  of  the  long  bones  is  transformed  into  a  soft,  bright  red  lymphoid 
tissue,  smears  from  which  show  great  numbers  of  megaloblasts.  Areas 
of  fatty  degeneration  are  characteristic,  especially  the  tiger-lily  spots 
in  the  heart  muscle.  Iron-containing  pigment  (hemosiderin)  is  found 
in  the  liver,  spleen,  and  kidneys.  Areas  of  degeneration  in  the  spinal 
cord  may  account  for  nervous  symptoms.  The  red  cells  frequently 
fall  below  2,000,000  with  patients  going  about.  Cases  have  been  re- 
ported with  counts  under  200,000.  The  color  index  is  high.  Megalo- 
blasts are  the  most  characteristic  qualitative  change  in  the  red  cells. 
Megaloblastic  crises  may  at  certain  times  show  enormous  numbers 
of  megaloblasts.  Cases  often  present  remissions  in  which  no  megalo- 
blasts can  be  found.  In  such  cases  the  presence  of  many  macrocytes 


SECONDARY   ANAEMIAS  2OI 

should  prevent  an  examiner's  reporting  against  a  pernicious  anaemia 
previously  diagnosed. 

Poikilocytosis,  polychromatophilia,  and  stippling  are  also  features 
of  the  disease.  Normoblasts  are  far  less  frequent  than  megaloblasts 
and  there  is  usually  a  moderate  lymphocytosis.  Myelocytes  may  be 
present,  but  their  precursors,  the  myeloblasts,  are  probably  more  fre- 
quently met  with. 

Cases  of  pernicious  anaemia  show  remissions  during  which  the  patient  is  ap- 
parently on  the  road  to  recovery.  Such  improvements  are  only  temporary.  The 
remissions  may  last  from  two  months  to  possibly  three  or  four  years.  Especially 
in  the  anaemia  of  Dibothriocephalus  latus  do  we  have  a  picture  of  pernicious  anae- 
mia. It  is  supposed  to  be  due  to  a  toxin  present  in  the  heads  of  these  tape-worms. 

Blood  changes  more  or  less  like  those  of  pernicious  anaemia  have  at  times  been 
noted  in  children  with  tuberculosis  of  bovine  nature.  The  human  strain  of  T.B. 
does  not  seem  to  produce  such  changes. 

An  acute  disease  showing  a  rapidly  developing  anaemia  of  the  pernicious  anaemia 
type  is  verruga  peruana  in  which  the  bone,  marrow  seems  especially  involved. 

SECONDARY  ANAEMIAS. 

These  are  the  anaemias  which  can  be  definitely  traced  to  some  dis- 
ease not  of  the  haemopoietic  system. 

There  are  two  main  groups — those  following  haemorrhage  and  those 
secondary  to  various  diseases.  If  the  haemorrhage  is  sudden  and  great, 
the  resulting  condition  is  one  of  oligochromaemia — chlorotic  in  type. 
Normoblasts  are  usually  found  after  the  third  day. 

The  low  Hb.  percentage  is  apt  to  continue  for  several  weeks.  There  is  also  an 
increase  in  the  percentage  of  polymorphonuclears. 

It  is  a  question  whether  prolonged  operation  or  those  requiring  narcosis  are 
justified  where  the  reduction  in  Hb.  is  under  40%.  (According  to  Miculicz,  30% 
is  the  minimum). 

Where  the  loss  of  blood  is  gradual,  as  in  gastric  cancer  or  severe  haemorrhoids 
the  picture  may  more  nearly  approach  that  of  pernicious  anaemia.  Secondary 
anaemias  usually  show  a  moderate  leukocytosis.  In  chronic  nephritis  and  prolonged 
suppurative  conditions  normoblasts  and  macrocytes  are  rare — moderate  poikilo- 
cytosis  with  the  presence  of  many  microcytes  being  the  rule. 

In  fatal  anaemia  from  chronic  acetanilide  poisoning  high  color  index,  macrocytes 
and  megaloblasts  have  been  noted. 

In  some  secondary  anaemias,  as  in  syphilis,  carcinoma,  and  tuber- 
culosis, we  have  a  chlorotic  color  index  (chloro-anaemias). 

In  secondary  anaemias  polychromatophilia,  poikilocytosis,  and  punc- 
tate basophilia  (stippling  )may  be  present.  This  latter  is  very  marked 


202  NORMAL   AND   PATHOLOGICAL  BLOOD 

in  lead  poisoning,  but  in  certain  cases  of  malarial  cachexia  it  may  be 
equally  prominent.  The  only  form  of  nucleated  red  cell  seen  is  the 
normoblast,  in  very  small  numbers,  or  it  may  not  be  present. 

Megaloblasts  are  practically  never  seen,  except  in  some  of  the  very  severe  para- 
sitic anaemias,  as  the  broad  Russian  tape-worm  infection.  The  red  cells  generally 
number  between  2,000,000  and  4,000,000,  thus  differentiating  chlorosis.  The 
leukocytes  are  frequently  increased  to  15,000.  In  the  anaemia  of  splenic  anaemia 
there  is  a  marked  leukopenia.  In  anaemias  from  malignant  tumors  the  color  index 
is  usually  of  the  chlorotic  type — the  haemoglobin  content  of  the  red  cells  being 
more  affected  than  the  number.  Normoblasts  are  usually  present,  and  this  find- 
ing may  differentiate  gastric  cancer  from  ulcer.  In  bone  marrow  metastases 
megaloblasts  may  be  expected.  Myelocytes  and  so-called  tumor  cells  (large  cells 
with  faintly-staining  vacuolated  nuclei  and  frut  little  cytoplasm)  may  also  be  found. 
As  a  rule,  there  is  a  moderate  leukocytosis  in  malignant  disease.  Eosinophiles 
may  be  largely  increased  in  sarcoma. 

THE  LEUKAEMIAS. 

It  is  in  the  leukaemias  that  we  have  the  greatest  increase  in  the  num- 
ber of  white  cells.  These  cases  show  more  or  less  anaemia,  but  we  may 
have  cases  of  myelogenous  leukaemia  showing  250,000  leukocytes  per 
cubic  millimeter  without  particular  change  in  the  red  cells.  The 'more 
marked  the  red-cell  change  the  more  severe  the  condition. 

There  are  two  well-defined  types  of  leukaemia,  the  lymphatic  and  the 
splenomyelogenous.  It  must  be  borne  in  mind,  however,  that  while 
a  greater  change  in  the  lymphatic  glands  may  produce  the  lymphatic 
type,  yet  even  in  such  cases  we  expect  to  find  alteration  in  bone  marrow 
and  spleen;  that  is,  there  is  a  general  involvement  of  the  haemopoietic 
system  in  all  leukaemias,  the  activity  being  most  marked  in  spleen  and 
bone  marrow  in  certain  cases  and  in  lymphatic  glands  in  others. 

Myelogenous  leukaemia  is  a  very  rare  disease,  about  five  times  as 
rare  as  pernicious  anaemia.  Lymphoid  leukaemia  is  still  more  rare. 

Splenomyelogenous  Leukaemia  (myeloid  leukaemia). — The  differen- 
tiation of  the  blood  picture  of  this  disease  from  leukocytosis  does  not 
depend  on  the  number  of  leukocytes,  but  on  the  presence  and  large 
proportion  of  myelocytes.  We  expect  both  neutrophilic  and  eosino- 
philic  myelocytes  in  myeloid  leukaemia — the  proportion  of  these  varies, 
but,  as  a  rule,  the  neutrophilic  one  is  the  common  one.  The  blood  in 
advanced  cases  is  milky  and  shows  a  most  marked  buffy  coat.  The 
marrow  is  largely  replaced  by  a  yellow  pyoid  material.  The  spleen 
may  weigh  10  pounds. 


THE    LEUKAEMIAS 


203 


The  leukocyte  count  is  on  the  average  from  200,000  to  500,000.  Cases  are 
reported  of  more  than  1,000,000  white  cells.  The  neutrophilic  myelocytes  make 
up  about  30  to  40%  of  these  and,  about  equal  in  number,  are  found  the  polymor- 
phonuclears,  while  the  percentage  of  the  lymphocytes  is  decreased  (2  to  5%)  and 
normal  eosinophiles,  eosinophilic  myelocytes,  and  large  mononuclears  make  up  the 
remaining  percentages.  We  usually  have  great  numbers  of  normoblasts.  Megal- 
oblasts  may  be  rarely  found.  The  red  count  is  usually  about  2,500,000  and  the 
color  index  low. 

Lymphatic  Leukaemia. — -In  this  we  have  glandular  enlargements, 
but  not  such  large  masses  as  in  Hodgkin's  disease.  The  red  cells  are 
usually  reduced  about  one-half  and  the  color  index  is  a  little  below 
normal.  Normoblasts  are  rarely  found.  Myelocytes,  as  a  rule,  are 


FIG.  56. — Myelogenous  leukaemia,     m,  Myelocyte;  p,  polymorphonuclear;  b,  mast 
cell;  n,  normoblast.     (Cabot.) 

absent,  but  may  amount  to  5%  of  the  leukocytes.  The  predominating 
leukocyte  (75  to  98%)  is  the  small  lymphocyte.  In  acute  lymphatic 
leukaemia  the  large  lymphocytes  predominate. 

These  however  are  pathological  and  differ  from  the  large  lymphocyte  in  not 
having  azur  granules  and  the  nucleus  stains  poorly  and  is  often  indented.  The 
leukocyte  count  is  never  so  great  as  in  myeloid  leukaemia,  rarely  exceeding  125,000. 

Pseudoleukaemia.— Hodgkin's  disease  is  usually  considered  as  a 
disease  with  marked  glandular  enlargements,  but  with  a  negative  blood 
picture,  or  at  any  rate  only  a  moderate  leukocytosis  with  a  relative 
increase  of  lymphocytes. 

The  red  cells  are  usually  above  3,000,000.  It  has  been  considered 
that  an  increased  percentage  of  transitionals  (10  to  15%),  should  a 
leukopenia  coexist,  is^characteristic. 


204  NORMAL  AND   PATHOLOGICAL  BLOOD 

Undoubtedly  the  view  that  so-called  lymphosarcomata,  lymphatic 
leukaemia,  and  Hodgkin's  disease  merge  into  one  another  and  that  they 
represent  a  malignant  cell  formation  in  the  haemopoietic  system  is  the 
conservative  one  to  take. 

A  certain  proportion  of  cases  of  Hodgkin's  disease,  however,  show 
endothelial  proliferation  and  a  chronic  fibroid  change. 

In  Kundrat's  lymphosarcoma  we  have  a  neutrophile  leukocytosis 
and  a  diminution  of  the  lymphocytes.  The  spleen  and  liver  are  rarely 
involved. 

Another  condition  with  swelling  of  the  lymphatic  glands,  which  do  not  how- 
ever fuse,  is  the  so-called  granulomatosis. 

In  this  we  have  a  polymorphonuclear  leukocytosis  of  from  20,000  to  50,000  with 


>• 


FIG.  57. — Lymphatic  leukaemia,     p,  polymorphonuclear;  m,  megaloblast;  e,  eosino- 
phile.     Twenty-one  lymphocytes  in  this  field.     (Cabot.) 

an  increase  in  the  percentage  of  eosinophiles.     The  lymphocytes  are  absolutely 
and  relatively  decreased.     In  granulomatosis  there  is  no  tendency  to  haemorrhage. 

Splenomegaly.— The  best  known  anaemia  associated  with  splenic 
enlargement  is  Banti's  disease. 

Banti's  disease  also  has  a  very  low  color  index  and  leukopenia.  In 
this  the  primary  affection  is  of  the  spleen  which  becomes  greatly  en- 
larged. The  accompanying  cirrhosis  of  the  liver  with  its  symptoms  of 
ascites,  etc.,  differentiate  it.  Splenectomy  often  cures  the  disease. 
The  leukopenia  is  one  showing  not  only  a  diminution  of  polymorphonu- 
clear percentage  but  of  cells  of  the  lymphocyte  type  as  well. 

There  is  a  considerable  increase  in  the  large  mononuclear  percentage.  Nu- 
cleated reds  and  myelocytes  are  invariably  absent.  It  must  be  remembered  that 


SPLENIC   ANAEMIA  205 

we  have  a  group  of  cases  showing  splenomegaly  which  are  syphilitic  in  origin  and 
which  as  a  rule  give  a  positive  Wassermann.  Clinically  or  haematologically  they 
resemble  true  Band's  disease  but  pathologically  the  spleen  shows  a  fibrosis  instead 
of  the  marked  increase  in  lymphatic  tissue  characteristic  of  Band's  disease. 

In  the  tropical  splenomegaly  or  kala  azar  we  have  a  marked  leukopenia  with 
a  marked  reduction  in  the  percentage  of  poly morphonu clears.  The  Gaucher  type 
of  splenic  anaemia  does  not  show  as  pronounced  and  early  an  anaemia  as  in  Band's 
type. 

Certain  conditions  which  partly  resemble  myelogenous  leukaemia 
and  partly  pernicious  anaemia  are  designated  leukanaemia.  Some  con- 
sider this  to  belong  to  the  group  of  diseases  in  which  the  multiple  mye- 
loma is  placed. 

In  splenomegalic  polycythaemia  we  have  a  red  count  of  from  9  to 
10  millions.  The  Hb.  percentage  may  be  200.  There  is  also  a 
leukocytosis  up  to  50,000.  Patients  are  cyanosed  and  have  a  very  large 
spleen. 

Splenic  anaemia  of  infancy  usually  occurs  between  the  ages  of  twelve 
and  twenty-four  months.  The  spleen  is  notably  enlarged  and  in  many 
cases  the  liver  is  equally  so.  The  red  cells  are  not  greatly  diminished 
in  number,  two  and  one-half  to  three  millions  being  usual  findings. 
Nucleated  reds  are  abundant.  While  a  leukocytosis  of  30,000  to  50,000 
is  often  present  it  is  markedly  less  than  that  of  splenomyelogenous 
leukaemia  and  the  increase  in  white  cells  is  more  of  those  of  lymphocyte 
type. 

The  color  index  is  very  low. 

Another  splenomegaly  of  children,  clinically  resembling  kala  azar,  is  caused 
by  Leishmania  infantum. 


NOTES  ON  BLOOD  WORK. 


NOTES  ON  BLOOD  WORK. 


NOTES  ON  BLOOD  WORK. 


NOTES  ON  BLOOD  WORK, 


NOTES  ON  BLOOD  WORK, 


PART  111. 
ANIMAL  PARASITOLOGY. 


CHAPTER  XV. 

GENERAL  CONSIDERATIONS  OF  CLASSIFICATION  AND 
METHODS. 

ANIMALS  that  are  in  all  respects  alike  we  term  a  Species.  Of  course 
the  male  and  female  of  a  species  may  be  very  unlike,  but  as  a  result  of 
mating  they  produce  young  having  characteristics  similar  to  the  parents. 
Now,  if,  as  in  the  case  of  the  mosquitoes  causing  yellow  fever,  we  find 
some  with  straight  silvery  lines  and  others  uniformly  showing  crescentic 
silveiy  bands  about  thorax,  yet  resembling  each  other  closely  in  the 
respect  of  being  dark,  brilliantly  marked  mosquitoes,  we  should  con- 
sider them  as  being  separate  species  with  a  certain  relationship  to  which 
the  term  Genus  is  applied. 

The  term  "genus"  is  of  wider  application  than  the  word  " species." 
Thus  animals  which  agree  in  the  main  characteristics  of  size,  proportion 
of  parts,  and  general  structure  are  placed  in  the  same  genus. 

In  naming  a  species  we  always  first  write  the  name  of  the  genus 
which  has  a  Greek  or  Latin  name,  commencing  with  a  capital,  and  follow 
with  the  specific  term,  which  latter  commences  with  a  small  letter. 
Thus  we  designate  the  dark  silver-marked  mosquitoes  as  belonging  to 
the  genus  Stegomyia;  those  showing  the  characteristics  of  curved  silver 
bands  and  two  central  parallel  lines  (lyre  pattern)  on  dorsal  surface  of 
thorax  we  designate  as  Stegomyia  calopus;  the  species  with  only  the 
straight  silver  lines  we  call  Stegomyia  scutellaris. 

If  the  specific  name  is  a  modern  patronymic  we  add  i  in  the  case  of  a  man  or 
ae  for  a  woman  to  the  exact  and  complete  name  of  the  person. 

Again,  certain  genera  show  resemblances  which  enable  us  to  make  broader 
groupings  to  which  we  apply  the  term  Subfamily.  Thus  the  genus  Stegomyia 
and  the  genus  Culex  have  the  similar  characteristics  of  palpi  in  the  female  being 
shorter  than  the  straight  proboscis;  we  therefore  classify  all  species  of  Stegomyia 

211 


212       CONSIDERATIONS   OF   CLASSIFICATION  AND   METHODS 

and  all  species  of  Culex  under  the  designation  Culicinae.  The  name  of  a  subfamily 
ends  in  "inse."  Now,  again,  certain  insects  are  different  from  others  in  having 
scales  on  the  wings.  We  find  that  not  only  do  the  Culicinae  have  such  character- 
istics; but  the  same  is  observed  with  the  Anophelinae  and  other  similar  scale-wing 
insects.  All  of  these  we  term  a  Family  and  we  speak  of  the  Culicidae,  meaning 
the  family  of  mosquitoes.  The  name  of  a  family  ends  in  "idae."  Many  families 
are  not  subdivided  into  subfamilies,  but  are  directly  separated  into  genera.  Again, 
a  genus  may  have  only  a  single  species. 

At  times  a  family  may  be  raised  to  superfamily  rank — the  subfamilies  then 
becoming  families.  Thus  the  families  Ixodidae  and  Argasidae  belong  to  the  super- 
family  Ixodoidea.  The  termination  for  a  superfamily  is  oidea. 

When  there  are  a  number  of  families  agreeing  closely  in  some  striking  character- 
istic, we  group  them  together  into  an  Order;  thus,  the  family  of  mosquitoes  closely 
resembling  many  other  families  of  insects  in  possessing  a  pair  of  well-developed 
wings  are  grouped  in  the  order  Diptera;  all  of  which  resemble  certain  other  animals 
in  the  possession  of  a  distinct  head,  thorax  and  abdomen  with  three  pairs  of  legs 
projecting  from  the  thorax.  This  collection  of  animals  we  call  a  Class;  thus,  we 
speak  of  the  class  Insecta.  It  will  be  observed  that  the  insects  have  no  internal 
skeleton,  but  instead  a  chitinous  cuticle,  the  exoskeleton.  Spiders,  ticks,  etc., 
resemble  them  in  this  respect,  and  we  now  apply  to  all  such  animals  the  wider 
designation,  Branch  or  Phylum  Arthropoda. 

Inasmuch  as  the  animal  kingdom  is  divided  into  the  branches  Protozoa,  Pori- 
fera,  Ccelenterata,  Echinodermata,  Vermes,  Arthropoda,  Mollusca  and  Chordata, 
we  see  that  the  branch  is  the  largest  grouping  we  employ.  To  descend  in  the  scale 
we  have  belonging  to  the  branch,  the  classes;  to  the  class,  the  orders;  to  the  order, 
the  families;  to  the  family,  the  subfamilies;  to  the  subfamily,  the  genera;  to  the 
genus,  the  species.  Occasionally  a  species  is  further  divided  into  subspecies. 

By  a  type  species  we  understand  the  species  of  a  genus  always  re- 
ferred to  as  representing  the  genus. 

While  other  species  of  a  genus  may  for  good  reason  be  transferred  to  another 
genus  the  type  species  is  permanently  in  the  genus.  Many  favor  alliteration  for 
type  species,  as  Heterophyes  heterophyes.  When  a  species  is  transferred  to  a  new 
genus  the  specific  name  goes  with  it. 

The  male  animal  is  designated  by  the  sign  of  Mars  (cf ),  the  female  by  that  of 
Venus  (9). 

There  are  certain  terms  employed  in  animal  parasitology  which  it  is  necessary 
to  understand.  Among  these  we  shall  refer  to  the  following: 

1.  True  Parasitism. — By  this  is  understood  the  condition  where  the  parasite 
does  harm  to  the  host,  deriving  all  the  benefit  of  the  association.     A  good  example 
of  this  would  be  the  hookworm  infecting  man  or  animals. 

2.  Mutualism. — In  such  an  association  there  is  mutual  benefit  to  each  party 
of  the  association.     An  instance  of  this  would  be  the  presence  of  colon  bacilli  in 
the  intestines.     The  bacillus  is  furnished  a  suitable  habitat  and  in  return  protects 
its  hDst  against  strictly  pathogenic  bacteria. 

Another  example  would  be  the  oyster  crab  found  inside  the  oyster  shell. 


ZOOLOGICAL  NOMENCLATURE  213 

3.  Commensalism. — Here  there  is  benefit  to  the  parasite,  but  no  injury  to  the 
host.  An  example  of  this  kind  would  be  furnished  in  the  case  of  the  Trichomonas 
vaginalis  which  lives  in  the  vaginal  mucus,  but  so  far  as  known,  does  no  injury  to 
the  host. 

If  the  Entamceba  coli  be  nonpathogenic  this  would  be  another  example. 

4.  Nomenclature.— When  the  thousands  of  different  species,  genera, 
etc.,  of  animals  is  considered,  it  will  be  readily  perceived  that,  unless 
some  system  existed  for  theii  designation,  indescribable  confvsion 
would  prevail.  To  avoid  this,  the  International  Code,  based  on  the 
rules  of  Linnaeus  (tenth  edition  of  Systema  naturae,  1758,  is  basis  of 
binary  zoological  nomenclature),  requires  Latin  or  Latinized  names. 

In  printed  matter  the  zoological  name  should  be  in  italics,  that  of  the  family 
in  Roman  type.  The  name  of  the  author  of  a  specific  name  is  written  immediately 
after  the  name  without  punctuation  and  may  be  followed  by  the  year  of  publica- 
tion set  off  by  a  comma,  thus:  Ascaris  lumbricoides  Linnaeus,  1758.  Should  the 
name  of  the  author  appear  in  parentheses  it  indicates  that  he  proposed  the  specific 
name  but  placed  the  species  in  another  genus  than  that  in  which  it  now  appears, 
and  the  name  of  the  author  responsible  for  placing  the  species  in  the  present  genus 
may  be  written  after  the  name  of  the  original  author  of  the  species;  for  example, 
Davainea  madagascariensis  (Davaine,  1869)  Blanchard,  1891,  tells  us  that  Davaine 
proposed  the  specific  name  madagascariensis  in  1869  but  placed  it  in  some  other 
genus  and  that  Blanchard  in  1891  transferred  it  to  the  genus  Davainea.  There 
are  certain  rules  governing  the  naming  of  animals.  Of  these,  the  law  of  priority 
provides  that  the  oldest  published  name,  under  the  code,  of  any  genus  or  species 
is  its  proper  zoological  name.  The  history  of  the  naming  of  the  organism  of  syphilis 
illustrates  this  well.  Schaudinn  gave  this  organism  in  1905  the  name  of  Spiro- 
chaeta  pallida.  Ehrenburg,  in  1838,  had  used  the  name  Spirochaeta  for  animals 
of  a  different  character,  so  that  this  designation  of  the  genus  was  not  permissible 
under  the  code.  Villemin,  a  little  later,  proposed  the  generic  name  Spironema. 
This  term,  however,  was  found  to  have  been  used  in  1864  by  Meek  for  a  genus  of 
molluscs  and  by  Klebs  in  1892  for  a  genus  of  flagellates.  Consequently,  being  a 
homonym,  it  was  not  available. 

(A  generic  name  can  be  applied  to  only  one  animal  genus  and  if  a  similar  name 
is  subsequently  given  another  genus  it  is  a  homonym  and  is  to  be  rejected.) 

On  December  2,  1905  Stiles  and  Pfender  then  proposed  the  name  Microspiro- 
nema,  but  as  Schaudinn  published  on  Oct.  26,  1905  the  designation  Treponema, 
the  name  Treponema  pallidum  had  to  be  accepted  as  the  proper  zoological  name 
for  the  organism  of  syphilis. 

Of  unusual  interest  is  the  question  of  the  name  of  the  old-world  hookworm. 
Dubini,  in  1843,  named  a  nematode  found  by  him  in  man  Agchylostoma.  By  the 
law  of  priority  this  spelling  would  have  been  the  correct  one  had  he  not  stated  in 
a  footnote  that  the  generic  name  was  derived  from  two  Greek  words  a-f-yvkoo  and 
orofia.  Having  indicated  the  origin  of  the  name  it  became  subject  to  the  rules 
for  correct  transliteration,  which  is  Ancylostoma. 


214      CONSIDERATIONS    OF    CLASSIFICATION   AND    METHODS 

In  case  of  larva  and  adult  or  male  and  female,  formerly  considered  different 
animals  but  subsequently  found  to  be  the  same,  the  oldest  available  name  becomes 
the  name  of  the  species. 

Another  point  is  that  names  are  not  definitions,  consequently  the  fact  of  lack 
of  appropriateness  of  any  name  is  no  objection  to  its  continuation.  This  will  ap- 
peal to  anyone  as  a  wise  provision,  because  if  a  different  name  were  substituted 
each  time  a  designation  more  descriptive  or  applicable  was  invented  it  would  be 
utterly  destructive  to  system.  When  it  is  considered  that  some  of  our  parasites 
have  approximately  fifty  different  designations,  for  the  most  part  given  by  med- 
ical observers,  it  will  be  appreciated  how  much  the  zoologist  has  aided  us  in  trying 
to  eliminate  all  but  the  single  proper  zoological  name. 

The  objections  so  frequently  heard  among  physicians  in  connection  with  adopt- 
ing new  names  for  old  ones  are  not  well  founded.  Wherever  confusion  has  reigned, 
the  establishment  of  order  always  results  in  temporary  greater  confusion.  There 
is  no  doubt  that  the  student  taking  up  this  subject  a  few  years  hence  will  have  the 
satisfaction,  thanks  to  the  zoologist,  of  only  having  to  burden  his  mind  with  one 
name  for  one  parasite. 

There  is  only  one  correct  name  for  an  animal  and  all  other  names 
are  synonyms. 

The  principal  cause  of  changes  of  names  is  that  our  conception  of  the  relation- 
ships of  animals  changes. 

5.  Terminology. — This  applies  to  appropriate  designations  for  different  organs, 
symptoms,  etc.,  and  is  not  subject  to  any  rule  other  than  that  of  good  usage. 

Thus  the  terms  cirrus  in  the  case  of  the  male  copulatory  organ  of  flukes,  spicule 
for  the  same  in  nematodes  and  penis  in  connection  with  insects  would  be  instances 
of  terminology. 

6.  Pseudoparasitism. — Where  organisms  enter  the  body  accidentally  and  when 
such  sojourn  in  the  body  of  man  plays  no  part  in  the  life  history  of  the  organism 
we  employ  the  term  pseudoparasitism.     For  example:     Fly  larvae  swallowed  by 
man  and  passed  out  in  the  faeces.     We  also  use  the  terms  temporary  parasites 
(bedbug)  and  permanent  parasites  (liver  fluke). 

7.  Hosts. — The  animal  in  which  a  parasite  undergoes  its  sexual  life  is  called  the 
definitive  or  final  host,  that  in  which  it  passes  its  larval  existence  the  intermediary 
host.     For  example:  Man  is  the  intermediary  host  of  the  malarial  parasite,  the 
mosquito  the  definitive  host.     A  single  animal  may,  however,  be  both  definitive 
and  intermediary  host;  thus  Trichinella  may  pass  its  larval  existence  in  the  muscles 
of  man  and  its  sexual  life  in  his  intestines. 

8.  Heredity,  Congenitalism. — Hereditary  characteristics  are  those  which  were 
present  in  the  ovum  or  spermatozoon  before  fertilization;  congenital  ones  those 
which  occur  after  fertilization.     South  African  tick  fever  is  probably  an  instance 
of  heredity,  the  spirochaetes  having  been  found  in  the  ovary  and  ova  of  the  female 
tick. 

9.  Heterogenesis,  Parthenogenesis. — Offspring  differs  from  parent,  but  after 
one  or  more  generations  there  is  reversion  to  the  parent  form. 

Strictly  speaking  the  term  heterogony  applies  to  reproduction  when  a  sexual 


PARTHENOGENESIS  215 

generation  alternates  with  a  parthenogenetic  one.  Where  a  nonsexual  genera- 
tion, as  by  division  or  budding,  alternates  with  a  sexual  one  the  process  is  called 
metagenesis.  In  parthenogenesis  reproduction  eggs  develop  without  the  occurrence 
of  fertilization  by  spermatozoa. 

In  coccidiosis  we  have  a  sexual  cycle  (sporogony)  alternating  with  a  nonsexual 
one  (schizogony).  In  the  infection  with  Strongyloides  we  have  a  sexual  cycle 
alternating  with  a  parthenogenetic  one.  In  malaria  we  have  a  sexual  generation, 
a  nonsexual  one  and  according  to  Schaudinn,  a  parthenogenetic  one,  which  latter 
accounts  for  malarial  relapses. 


CHAPTER  XVI. 


THE  PROTOZOA. 

CLASSIFICATION   OF  PROTOZOA. 


Class 


Order 


Rhizopoda  Gymnamoeba 

(Sarcodina) 

These  throw  out  protoplas- 
mic projections  called  pseudo- 
podia. 


Flagellata 

(Mastigophora) 
These  move  by  means  of 
undulating     membranes     or 
flagella. 


Infusoria  Heterotricha 

(Ciliata) 

These  have  contractile  vacu- 
oles  and  numerous  fine  cilia 
which  are  shorter  than  flagella 
and  have  a  sweeping  stroke. 

Sporozoa 

These  have  no  motile  organs. 
They  live  parasitically  in  the 
cells  or  tissues  of  otlrer  animals. 
Reproduction  by  spores. 


Genus 


Entamoeba 


Leydenia 


Species 
E.  coli 

E.  histolytica 
E.  tetragena 
E.  buccalis 


L.  gemmipara 


(S.  recurrentis 

S.  vincenti 

S.  duttoni 

S.  carteri 

S.  refringens 

Schizotrypanum          S.  cruzi 

Treponema 

'  T.  pallidum 
T.  pertenue 

Trypanosoma 

T.  gambiense 
T.  rhodesiense 

Trichomonas 

T.  vaginalis 
T.  intestinalis 

Lamblia                       L-  intestinalis 

Babesia                         B.  bigemina 

{L.  donovani 

L.  tropica 

L.  infantum 

Balantidium                B.  coli 

Coccidiaria 


Eimeria 
Isospora 


Haemosporidia       Plasmodium 


E.  stiedae 
I.  bigemina 

P.  vivax 
P.  malarise 
P.  falciparum 


2l6 


BINUCLEATA  217 

NOTE. — Hartmann  and  others  have  grouped  the  Haemosporozoa  and  the  Haemo- 
flagellata  in  an  order  BINUCLEATA.  The  main  characteristic  is  the  possession  of 
two  differentiated  nuclei,  the  kinetonucleus  and  the  trophonucleus,  at  some  develop- 
mental or  transitional  stage.  While  trypanosomes  plainly  show  these  characteris- 
tics certain  others,  as  the  malarial  parasites  and  the  leishman-donovan  bodies,  hav- 
ing been  modified  as  the  result  of  cell  parasitism,  do  not  do  so.  This  grouping  to- 
gether of  the  blood  flagellates  and  sporozoa  under  the  name  Binucleata  has  been  con- 
sidered by  many  protozoologists  as  possibly  convenient  but  not  resting  on  sufficient 
ground  to  cause  organisms  with  similar  life  histories  as  Plasmodium  and  Coccidium 
to  be  separated  and  the  former  to  be  placed  with  the  blood  flagellates  in  a  new 
grouping. 

THE  PROTOZOA. 

By  the  term  protozoa  we  understand  a  branch  of  animals  in  which  a 
single  cell  is  morphologically  and  functionally  complete;  it  is  not  one  of 
a  number  of  cells  going  to  make  up  a  complex  individual  and  dependent 
on  such  a  combination  as  is  the  case  with  the  metazoa  (there  is  no 
differentiation  into  tissues  in  protozoa). 

Recognizing  the  fact  that  certain  protozoa  have  characteristics  which  make 
it  impossible  to  draw  a  distinction  between  them  and  plants  Haeckel  has  proposed 
the  name  Protista  as  a  designation  for  all  simple  and  primitive  living  organisms 
whether  they  be  plants  or  animals.  In  such  a  classification  we  would  have  the 
kingdom  of  Protista  as  well  as  the  animal  and  vegetable  kingdoms.  In  such  a 
grouping  the  bacteria  would  be  the  lower  types  and  the  fungi  and  protozoal  organ- 
isms the  higher  ones. 

The  protozoal  cells  are  made  up  of  protoplasm  which  is  divided  into  nucleus 
and  cytoplasm.  The  cytoplasm  is  at  times  separated  into  an  external,  hyaline 
portion,  the  ectoplasm  or  ectosarc  and  an  internal  granular  portion,  the  endo- 
plasm  or  endosarc.  The  functions  of  the  ectosarc  are  protective,  locomotor,  ex- 
cretory and  sensory;  those  of  the  endosarc  trophic  and  reproductive.  Protozoa 
may  be  holozoic  (animal  like)  or  holophytic  (plant  like),  saprophytic  (fungus 
like),  or  parasitic  (living  at  the  expense  of  some  other  animal  or  plant). 

The  nucleus  is  characterized  by  concentration  of  the  so-called  chromatin  sub- 
stance of  the  cell.  This  chromatin  however  is  usually  combined  with  achromatin. 
The  usually  accepted  test  for  chromatin  is  the  staining  affinity  for  basic  aniline 
dyes.  This  test  is  now  known  to  be  unsatisfactory  as  other  substances  than*  chro- 
matin may  stain  even  more  intensely.  When  chromatin  is  scattered  through  the 
cytoplasm,  as  extranuclear  aggregations,  such  chromatin  granules  are  called  chro- 
midia.  There  are  cells  where  the  chromidia  take  the  place  of  the  nucleus  and  from 
which  a  nucleus  may  be  formed.  Chromidia  may  arise  from  nuclei  and  nuclei 
from  chromidia.  The  nucleus  is  made  up  of  a  network  of  linin  in  which  achro- 
matic reticulum  is  contained  the  nuclear  sap  or  karyolymph.  As  a  rule  an  achro- 
matic nuclear  membrane,  continuous  with  the  reticulum,  separates  the  nucleus 
from  the  cytoplasm.  In  addition  we  have  a  substance  which  is  achromatic  (plas- 
tin)  and  which  is  the  imbedding  substance  for  chromatin  grains.  These  plastin 
chromatin  combinations  are  called  karyosomes.  The  nucleoli  are  probably  pure 
plastin.  Plastin  is  to  be  regarded  as  a  secretion  or  modification  of  chromatin 


2l8  THE   PROTOZOA 

made  to  serve  as  a  matrix  for  the  chromatin.  Chromatin  may  be  concentrated 
in  a  single  mass  so  that  the  nuclear  space  looks  like  a  vesicle  with  a  central  chroma- 
tin  mass  (vesicular  nucleus)  or  numerous  chromatin  grains  may  be  scattered  through 
the  nuclear  space  (granular  nucleus).  The  centrosome,  which  presides  over  cell 
division,  is  usually  located  just  outside  the  nucleus.  In  some  protozoa  however 
the  centrosome  is  within  the  nucleus  and  is  often  seen  inside  of  a  karyosome  and  is 
then  called  a  centriole.  The  centrosome  may  also  function  over  kinetic  activities 
(flagellar  motion)  and  is  then  termed  blepharoplast. 

Certain  protozoa,  as  trypanosomes,  show  a  differentiation  of  nuclei,  the  larger 
trophonucleus  governing  the  functions  of  general  metabolism  and  the  smaller  kine- 
tonucleus  directing  the  motor  activities.  Infusoria  have  a  larger  macronucleus 
which  contains  vegetative  chromatin  and  a  smaller  micronucleus  which  contains 
reserve  reproductive  chromatin. 

Reproduction  of  protozoa  may  be  by  fission,  when  the  nucleus  and  cytoplasm 
divide  into  two  by  simple  division. 

When  the  nuclei  divide  into  a  number  of  daughter  nuclei,  which  is  followed  by 
multiple  division  of  the  cytoplasm,  we  have  sporulation. 

Instead  of  fission  we  may  have  sexual  reproduction  or  conjugation  (zygosis). 
Here  the  nuclei  of  the  separate  sexual  individuals  (gametes)  are  termed  pronuclei 
and  the  product  of  their  fusion  a  synkaryon. 

Where  a  single  cell  has  division  of  its  nucleus  with  subsequent  fusion  of  these 
daughter  nuclei  to  form  a  synkaryon  the  process  is  termed  autogamy. 

If  two  similar  cells  conjugate  the  term  is  isogamy;  if  dissimilar  as  the  macro- 
gametes  and  microgametes  of  malaria,  anisogamy. 

The  process  of  sexual  union  is  termed  syngamy  and  is  of  two  kinds  (i)  when 
the  two  gametes  fuse  completely  or  copulation  and  (2)  when  they  remain  separate 
and  only  exchange  nuclear  material  or  conjugation. 

The  structures  of  protozoa  concerned  in  movement,  metabolism,  etc.,  are  termed 
organelles.  Of  the  former,  pseudopodia,  flagella,  cilia  and  myonemes  (contractile 
fibrils  which  give  support  to  the  body  cell  of  certain  protozoa)  may  be  given  and 
food  vacuoles  and  contractile  vacuoles  of  the  latter.  The  contractile  vacuole 
which  is  probably  an  excretory  organelle  is  absent  in  almost  all  parasitic  protozoa. 
It  is  however  present  in  ciliates. 

RHIZOPODA  (SARCODINA). 

In  this  class  of  protozoa  the  pseudopodia  serve  the  double  purpose 
of  nutrition  and  locomotion.  These  protoplasmic  extensions  may  be 
quite  broad  or  very  narrow — the  lobose  and  the  reticulose. 

As  a  rule,  the  thicker  the  pseudopod  the  more  rapid  the  movement. 
Some  rhizopods  have  hard  shell-like  coverings  which  are  secreted  in  or 
on  the  ectosarc.  These  skeletons  have  openings  through  which  the 
pseudopods  project.  The  pseudopodia  may  be  made  up  only  of  ecto- 
plasm or  both  ectoplasm  and  endoplasm  may  take  part.  Amoeboid 
movement  always  starts  in  the  ectoplasm.  In  addition  to  the  nucleus, 


THE   AMCEB^E   OF   MAN 


2IQ 


which  the  so-called  chromatin-staining  methods  bring  out  as  reddish 
areas,  we  frequently  observe  smaller  aggregations  of  chromatin-staining 
material  in  the  cytoplasm.  This  extranuclear  chromatin  is  supposed 
to  play  a  part  in  .the  more  intricate  divisions  which  such  protozoa  un- 
dergo. Food  vacuoles  and  contractile  vacuoles  are  present  in  many 
rhizopods. 

Entamoeba  coli  (Amoeba  coli). — This  is  considered  by  Schaudinn  to 
be  a  harmless  inhabitant  of  the  intestines  and  its  presence  in  the  faeces 
is  not  considered  of  importance. 


FIG.  58. — Various  protozoa,  i,  Entamoeba  coli;  2,  Entamceba  histolytica;  3, 
Leydenia  gemmipara;  4,  Trichomonas  vaginalis;  5,  Trichomonas  intestinalis;  6, 
Lamblia  intestinalis;  7,  flagellated  Leishmania  donovani;  8,  Leishmania  donovani 
in  phagocyte;  9,  Eimeria  stiedae;  10,  Isospora  bigemina;  n,  Trypanosoma gambiense; 
12,  Balantidium  coli. 

It  is  now  recognized  that  amoebae  of  man  are  not  cultivable.  When  we  obtain 
cultures  on  the  various  nutrient  poor  agar  plates,  formerly  so  much  used,  we  find 
that  the  amoebae  belong  chiefly  to  water  amoebae,  in  particular  a  Limax. 

The  only  safe  way  in  recognizing  amoebae  in  stools  is  to  note  amoeboid  movement. 
The  encysted  amcebae,  except  by  the  experienced,  can  scarcely  be  differentiated 
from  many  vegetable  cells  and  especially  from  large  phagocytic  cells,  of  probable 
endothelial  origin.  By  the  use  of  neutral  red  in  very  dilute  solution  the  granular 
endoplasm  will  be  observed  to  take  up  the  brick-red  stain. 


220  THE   PROTOZOA 

A  method  for  bringing  out  the  nuclear  features  is  as  follows:  take  a  loopful  of 
2%  acetic  acid  and  a  loopful  of  2%  formalin.  Tinge  the  mixture  to  a  rose  color 
with  neutral  red  and  then  stir  in  a  little  saturated  aqueous  solution  methyl  green, 
using  a  tooth  pick  which  has  been  dipped  into  the  methyl  green. 

In  staining  with  iron  haematoxylin  or  better  with  phosphotungstic  haematoxylin 
proper  fixation  is  very  important.  Fix  in  100  parts  of  sat.  aq.  sol.  bichloride  to 
which  is  added  50  c.c.  absolute  alcohol  and  5  drops  glacial  acetic  acid.  The  stain 
should  be  poured  on  the  moist  smear  of  faeces.  The  fixative  should  be  heated  to 
60°  C.  and  should  only  act  for  10  to  20  seconds.  Then  place  in  cold  sublimate 
alcohol  for  10  minutes  wash  in  70%  alcohol  colored  to  a  rich  port  wine  color  with 
iodine,  then  in  70%  alcohol,  then  in  water  and  then  stain  as  preferred.  Some  like 
a  carmine  stain. 

E.  coli  varies  greatly  in  size  (8  to  40^).  There  is  no  well-marked  distinction 
between  a  granular  interior  and  a  more  compact,  hyaline  exterior.  The  nucleus  is 
centrally  situated,  is  distinct,  and  on  staining  with  Wright's  stain  shows  the  chro- 
matin  coloration.  The  nucleus  is  rich  in  chromatin  and  with  iron  haematoxylin  it 
shows  four  chromatin  aggregations  lining  the  nuclear  membrane.  It  is  sluggishly 
motile  and  is  of  a  grayish-white  color.  When  stained  it  does  not  show  a  distinction 
between  endoplasm  and  ectosarc.  The  infecting  stage  is  an  encysted  form  with 
eight  nuclei  or  spores. 

Entamceba  histolytica  (Amoeba  dysenteriae).— This  is  considered 
the  pathogenic  amoeba.  Schaudinn  considered  that  it  was  by  the 
possession  of  its  tough,  tenacious  glassy,  and  highly  refractile  ectoplasm 
that  it  was  able  to  bore  its  way  into  the  submucosa  of  the  large  intestine 
and  bring  about  those  gelatinous-like  necroses,  which,  by  undermining, 
eventually  result  in  dysenteric  ulcerations. 

It  was  also  thought  to  be  the  species  found  in  tropical  liver  abscess. 
As  described  by  Schaudinn,  it  has  a  marked  differentiation  between  the 
glassy  ectoplasm  and  the  granular  endoplasm.  The  nucleus  is  indis- 
tinct, eccentric,  or  even  peripherally  situated,  and  stains  feebly. 

The  movement  is  more  active  and  the  color  more  greenish-yellow  than  E.  coli. 
Craig  notes  the  characteristic  staining  of  the  E.  histolytica,  this  being  a  dark  blue 
ectoplasm  encircling  a  lighter  blue  endoplasm.  In  dividing,  there  is  a  process  of 
budding.  These  little  spore-like  bodies  form  at  the  periphery  of  the  encysted  amoeba 
and  are  the  infecting  stage.  Faeces  should  be  examined  as  soon  as  possible  after 
the  stool  is  passed  in  order  that  one  may  have  the  best  opportunity  to  observe 
movement.  A  particle  of  mucus  pressed  down  with  a  cover-glass  makes  a  satis- 
factory preparation.  If  necessary  to  dilute,  use  blood-warm  salt  solution — not 
plain  water. 

Amoebae  were  first  described  by  Lambl  in  1859.  Found  by  Loesch  in  dysenteric 
stools  in  1875.  Councilman  and  Lafleur  in  1891  separated  amoebae  into  pathogenic 
and  nonpathogenic  strains. 

Kartutis  produced  dysentery  in  cats  by  introducing  dysenteric  stools  into  the 
rectum.  Kruse  and  Pasquale  produced  dysentery  with  liver  abscess  pus  which 


AMCEB.E 


221 


was  bacteriologically  sterile.  Shiga  in  1898  separated  the  bacillary  type  of  dysen- 
tery from  the  amoebic  one.  Schaudinn,  in  1903,  stated  that  E.  histolytica  was  the 
pathogenic  amoeba  of  man.  Viereck  found  encysted  amoebae  in  dysenteric  stools 
containing  four  nuclei.  This  amoeba  is  now  believed  to  be  the  common  pathogenic 
amoeba  of  man  and  is  named  E.  tetragena. 

Entamceba  tetragena. — This  amoeba  has  a  homogenous  and  highly 
refractile  ectoplasm  with  a  nucleus  richer  in  chromatin  than  E.  his- 
tolytica. It  has  a  central  karyosome  which  varies  in  size. 


FIG.  59. — Human  amoebae  showing  vegetative  and  encysted  stages.  Water 
amoebae  for  comparison.  (ia)  Entamceba  coli;  (16)  E.  coli  (encysted);  (2)  E. 
japonica;  (30)  E.  histolytica;  (36)  E.  histolytica  (encysted);  (30)  E.  histolytica  per- 
ipheral buds;  (40)  E.  tetragena;  (46)  E.  tetragena  (encysted);  (50)  water  amoeba, 
vegetative;  (56)  water  amoeba,  encysted. 


In  an  iron  haematoxylin  preparation  this  karyosome  shows  a  central  spot  or 
centriole  which  niay  fill  up  most  ol  the  nuclear  space  but  in  such  case  is  surrounded 
by  a  clear  zone  with  the  karyosome  ring  outside.  Hartmann  found  that  some  of 
Schaudinn's  specimens  were  E.  tetragena  and  the  belief  is  now  growing  that  the  life 
history  of  a  nucleus  resolving  into  chromidia  which  collected  at  the  periphery  and 
formed  the  peripheral  infecting  spores  was  an  error  in  observation  on  the  part  of 
Schaudinn  and  that  the  true  life  history  of  the  pathogenic  human  amoeba  is  that  of 
E.  tetragena.  In  such  case  E.  tetragena  and  E.  histolytica  applying  to  the  same 


222  THE   PROTOZOA 

amoeba  we  must  drop  the  name  E.  tetragena  by  reason  of  priority  of  E.  histolytica. 
Craig  now  takes  this  view. 

Wenyon  has  recently  produced  dysentery  in  kittens  by  infecting  them  with 
material  containing  the  four  spores  of  the  encysted  E.  tetragena.  He  also  produced 
liver  abscesses  in  one  of  the  kittens  experimentally  infected  with  dysentery. 

Entamoeba  buccalis. — This  has  an  ectoplasm  similar  to  E.  histolytica,  but  has  a 
centrally  situated  nucleus,  the  nucleus,  however,  is  poor  in  chromatin. 

Obtained  from  the  mouths  of  persons  with  dental  caries.  It  does  not  appear 
to  have  pathogenic  characteristics. 

Castellani  has  reported  an  intestinal  amoeba  with  an  undulatory  membrane. 
He  has  given  it  the  name  of  E.  undulans. 

Leydenia  gemmipara. — It  is  a  question  whether  these  bodies  were  animal 
parasites  or  simply  body  cells  showing  amoeboid  movement.  They  were  found 
in  the  ascitic  fluid  of  two  cases  of  carcinomatosis.  They  varied  in  size  from  3  to  36^. 

FLAGELLATA  (MASTIGOPHORA). 

In  this  class  of  protozoa  the  adults  have  flagella  for  the  purposes  of 
locomotion  and  the  obtaining  of  food. 

Some  flagellates  more  or  less  resemble  rhizopods  in  being  amoeboid  and  in  having 
an  ectoplasm  and  an  endoplasm.  The  body  is  frequently  covered  by  a  cuticle 
(periplast).  Some  flagellates  have  a  definite  mouth  part,  the  cytostome,  which  leads 
to  a  blind  oesophagus;  others  absorb  food  directly  through  the  body  wall.  In  addi- 
tion to  flagella,  some  flagellates  possess  an  undulating  membrane.  All  flagellates 
possess  a  nucleus  and  some  have  contractile  vacuoles.  The  flagellum  may  arise 
directly  from  the  nucleus  or  from  a  small  kinetic  nucleus,  the  blepharoplast  (micro- 
nucleus  or  basal  granule). 

The  most  important  flagellates  of  man  are  the  haemoflagellates.  Among  these 
we  may  include  the  blood  spirochaetes  and  the  organism  of  syphilis,  which  have 
many  resemblances  to  the  spiral  forms  of  bacteria,  together  with  the  three  genera  in 
which  protozoal  characteristics  are  marked,  namely,  Leishmania,  Trypanosoma 
and  Trypanoplasma.  In  addition  we  have  flagellates  in  the  intestinal  canal  and  in 
the  vaginal  secretion.  Some  authors  place  the  genus  Piroplasma  with  the  flagellates 
and  there  has  been  controversy  concerning  the  nature  of  certain  projections  from 
these  bodies.  It  would  seem  preferable,  however,  to  consider  them  under  the 
Sporozoa. 

Spirochaeta. 

The  generic  term  Spirochseta  is  applied  to  flagellates  having  a  spiral 
shape,  an  undulating  membrane,  and  no  flagella.  This  genus  is  one 
about  which  there  are  two  views :  one,  that  the  members  belong  to  the 
bacteria;  the  other,  that  they  are  protozoa.  The  absence  of  demonstra- 
ble nucleus  and  blepharoplast  makes  them  apparently  vegetable  in  nature 
while  the  variations  in  thickness,  the  fact  of  transmission  by  an  arthro- 


RELAPSING   FEVER  223 

pod,  and  indications  of  a  longitudinal,  rather  than  a  transverse  division, 
would  indicate  protozoal  affinities. 

It  would  seem  from  recent  investigations  that  both  methods  occur — longitudinal 
division  occurring  when  there  are  few  organisms  in  the  blood  and  transverse  at  the 
height  of  the  infection. 

Minchin  has  adopted  the  name  Spiroschaudinnia,  proposed  by  Sambon,  for  the 
parasitic  blood  spirochaetes 

S.  recurrentis. — This  is  the  organism  of  relapsing  fever.  It  was  formerly  con- 
sidered a  bacterium  and  was  termed  the  Spirochaeta  obermeieri  (discovered  by 
Obermeier  in  1873). 

It  is  present  in  the  blood  of  persons  suffering  from  the  disease  during  the  pyrexia. 
During  the  apyrexia  they  are  not  found  in  the  peripheral  circulation.  At  this  time 
they  are  present  in  great  numbers  in  the  spleen  where  they  are  actively  phagocytized. 


FIG.  60. — Spirochaetae  of  relapsing  fever  from  blood  of  a  man.     (Kolle  and 

Wassermann.} 

The  disease  is  supposed  to  be  transmitted  by  bedbugs  or  lice.  Monkeys  are  sus- 
ceptible and,  after  passage  of  the  organism  through  monkeys,  rats  can  be  infected. 

S.  duttoni.— This  is  the  cause  of  South  African  tick  fever  or  "  tete-fever. "  The 
disease  is  similar  to  relapsing  fever,  but  there  are  generally  four  or  five  febrile  par- 
oxysms with  apyrexial  intervals.  The  disease  is  readily  transmitted  to  ordinary 
laboratory  animals,  especially  the  rat. 

A  certain  degree  of  immunity  is  conferred  by  an  injection  with  a  certain  spiro- 
chaete,  but  this  does  not  hold  for  other  species;  thus,  rats  which  have  recovered 
from  S.  recurrentis  can  be  infected  by  S.  duttoni  and  vice  versa.  The  disease  is 
transmitted  by  the  bite  either  of  the  adult  or  larval  Ornithodoros  moubata.  Koch 
found  spirochaetes  in  the  eggs  of  the  ovaries  of  ticks  which  had  fed  on  persons  with 
the  disease.  It  is  thus  an  instance  of  hereditary  transmission. 

Leishman,  who  believes  in  the  protozoal  nature  of  these  organisms,  has  observed 
clumps  of  chromatin  granules  in  the  Malpighian  tubes  and  in  the  ovaries  of  infected 
ticks,  which  granules  he  considers  developmental  stages.  Material  showing  such 


224  THE   PROTOZOA 

granules  but  no  spirochaetes  has  brought  about  spirochaete  infection  in  mice.  He 
considers  that  infection  probably  occurs  through  material  voided  from  the  Mal- 
pighian  tubes  rather  than  through  the  medium  of  veneno-salivary  secretions. 

Other  spirochaetes  that  have  been  considered  as  pathogenic  for  the  type  of  re- 
lapsing fever  in  India  and  that  of  America  are  the  S.  carteri  and  the  S.  novyi. 

Nicolle  has  shown  with  relapsing  fever  of  Algiers  that  the  body  louse  can  trans- 
mit the  infection  by  spirochaete  containing  material  from  the  crushed  louse  being 
rubbed  into  the  wound  made  by  the  louse  in  biting.  Eggs  from  an  infected  louse 
hatch  out  infected  young  lice,  thus  showing  the  hereditary  transmission.  It  is 
now  also  considered  that  infection  with  South  African  relapsing  fever  by  O.  moubata 
occurs  by  the  rubbing  in  of  spirochaete  containing  faeces  into  the  wound  made  by 
the  bite  of  the  tick.  These,  as  with  plague  infection  from  the  contaminated  faeces 
of  the  rat  flea,  are  instances  of  the  contamination  mode  of  infection.  Noguchi  has 
recently  cultivated  the  various  species  of  pathogenic  human  spirochaetes  by  employ- 
ing a  method  similar  to  that  used  in  cultivating  the  organism  of  syphilis.  He  noted 
longitudinal  division  in  his  cultures. 

S.  vincenti. — This  is  a  very  delicate  spiral-shaped  organism  which  has  been  found 
in  conjunction  with  a  fusiform  bacillus  in  a  throat  inflammation,  usually  termed 
Vincent's  angina.  , 

S.  refringens. — This  Spirochaeta  is  frequently  associated  with  the  Treponema 
pallidum  and  is  common  in  genital  ulcerations.  It  is  thicker,  has  less  regular  and 
more  flattened  curves  and  stains  more  readily.  By  "dark  ground  illumination"  it 
is  thicker,  of  a  yellow  tint  instead  of  pure  white,  and  moves  in  its  entire  length. 

Treponema. 

The  genus  Treponema  has  no  undulating  membrane  and  has  a 
flagellum  at  each  end. 

Treponema  pallidum  (Spirochaeta  pallida). — -This  is  the  cause  of 
syphilis.  It  is  characterized  by  the  very  geometric  regularity  of  the 
spirals,  which  are  deeply  cut,  and  in  focusing  up  and  down  continue  in 
focus  (like  a  corkscrew).  They  require  about  thirty  minutes  to  stain 
distinctly  with  Giemsa's  stain  and  the  attenuated  ends  or  flagella  should 
always  be  noted  before  reporting  their  presence. 

Treponemata  are  found  in  the  cellular  areas  surrounding  the  thickened  blood- 
vessels and  in  the  coats  of  the  larger  arteries.  To  stain  them  in  section  Levaditi's 
method  is  the  best. 

The  India-ink  method  of  Burri  is  highly  recommended.  Take  one  loopful  of 
secretion  from  a  chancre  and  deposit  it  on  one  end  of  a  slide.  Surround  this  drop 
with  five  loopfuls  of  distilled  water  and  five  loopfuls  of  Giinther  and  Wagner's  ink. 
Mix  and  make  a  smear  as  for  blood.  When  dry  examine  with  the  oil  immersion 
objective  and  the  treponemata  will  be  found  to  stand  out  as  white  spirals  against 
a  dark  background.  Treponemata  often  appear  as  if  bent  in  the  middle. 

Harrison  prefers  collargol  to  India-ink.  One  part  of  collargol  is  put  in  a  bottle 
with  19  parts  of  water  and  well  shaken.  This  shaking  is  repeated.  One  loopful 


SYPHILIS 


225 


of  the  suspected  serum  and  one  loopful  of  the  collargol  suspension  are  mixed  and 
smeared  out  and  examined  as  for  the  India-ink  method. 

T.  pallidum  has  been  cultivated  anaerobically  in  horse  serum  by  Schereschewsky. 
The  cultures  contained  other  organisms.  Muhlens,  by  growing  anaerobically  on 
horse-serum  agar  (i  to  3),  claims  to  have  obtained  pure  cultures.  Animal  inocu- 
lations with  this  material  were  negative,  however. 


-AVERY- 


FIG.  6 1. — Binucleata,  (Haemoflagellata  and  Haemosporozoa).  i.  Schizo- 
trypanum  cruzi;  (a)  Merozoite  just  entering  r.b.c.;  (£)  fully  developed  trypanosome 
form  in  blood;  (c)  form  found  in  intestine  Conorhinus;  (d)  form  in  salivary 
gland  of  Conorhinus;  (e)  merocyte  from  the  schizogenous  cycle  in  lungs.  2.  Leish- 
mania  donovani;  (a)  Parasites  from  spleen  smear,  free  and  packed  in  phagocytic 
cell;  (b)  and  (c)  flagellate  forms  from  cultures.  3.  Trypanosoma  gambiense.  4. 
Plasmodium  vivax;  (a)  young  schizont;  (b)  uninfected  red  cell;  (c)  red  cell,  punctate 
basophilia;  (d)  merocyte;  (e)  macrogamete;  (/)  adult  schizont.  5.  Plasmodium 
malariae;  (a)  half-grown  schizont  showing  equatorial  band;  (b)  macrogamete;  (c) 
merocyte;  (d)  young  schizonts.  6.  Plasmodium  falciparum;  (a)  red  cell  showing 
multiple  infection;  (b)  young  ring  form;  (c)  crescent;  (d)  young  schizont  on  per- 
iphery of  r.b.c.  7.  (a)  Treponema  pallidum;  (b)  Spirochaeta  refringens.  8.  Trepon- 
ema  pertenue. 

Noguchi  has  cultivated  T.  pallidum  under  strict  anaerobic  conditions  in  a  medium 
of  ascitic  fluid  containing  a  piece  of  fresh  sterile  tissue,  preferably  placenta.  The 
growth  is  faintly  hazy  and  does  not  have  an  offensive  odor.  Spirochaeta  micro- 
dentium  shows  similar  morphology  but  the  cultures  have  a  foul  odor.  Sp.  macro- 
dentium  is  similar  culturally  but  differs  morphologically. 

When  cultures  of  T.  pallidum,  grown  for  one  or  more  weeks  in  ascitic  fluid  agar 
IS 


226  THE   PROTOZOA 

and  ascitic  fluid  are  ground  in  a  mortar,  heated  to  60°  C.  for  one  hour  then,  with  the 
final  addition  of  i%  trikresol,  we  have  an  emulsion  called  "luetin."  This  extract 
produces  an  allergic  reaction  on  the  skin  of  certain  syphilitics  (Luetin  reaction). 
To  carry  out  the  test  luetin  is  introduced  intradermally  at  the  insertion  of  the  left 
deltoid  and  a  control  emulsion  of  agar  media  injected  in  the  right  arm.  A  negative 
result  shows  as  an  erythema  without  pain  or  papule  formation.  Positive  reactions 
show  as  papules  vesicles  or  even  pustules  giving  rise  to  discomfort  for  several  days. 
While  the  control  side  usually  becomes  normal  in  forty-eight  hours  yet  in  latent 
and  tertiary  syphilis  the  control  may  show  almost  as  marked  a  reaction.  The  term 
"  Umstimmung  "  is  applied  to  this  susceptibility  to  trauma  of  the  skin  of  those  having 
tertiary  syphilis.  Some  cases  of  parasyphilitic  infections  which  are  negative  to  the 
Wassermann  test  give  a  positive  luetin  reaction. 

Noguchi  has  recently  demonstrated  T.  pallidum  in  all  layers  of  the  cerebral 
cortex  except  the  outermost  one  in  12  cases  out  of  70  cases  of  general  paresis  examined. 

In  diagnosis  either  use  the  dark  ground  illuminator  or  make  a  thin  smear  from 
the  sanious  oozing  after  vigorous  friction  of  the  chancre  with  gauze,  taking  up  this 
blood-stained  serum  on  the  end  of  a  slide  and  smearing  the  surface  of  a  second  slide 
with  the  adhering  material.  It  is  in  most  cases  more  satisfactory  to  curet  the  lesion, 
in  this  way  obtaining  material  from  the  areas  of  the  thickened  arteries. 

In  the  diagnosis  of  cerebrospinal  syphilis  we  use,  in  addition  to  the  Wassermann 
test  of  the  blood,  (i)  the  Nonne-Apelt  reaction  in  which  about  i  c.c.  of  a  saturated 
aqueous  solution  of  ammon.  sulphate  is  added  to  an  equal  amount  of  cerebrospinal 
fluid.  If  turbidity  or  rather  opalescence  appear  immediately,  or  within  three  minutes, 
the  test  is  positive.  (2)  The  counting  of  the  lymphocytes  in  the  cerebrospinal 
fluid.  A  lymphocytosis  occurs  in  cerebrospinal  syphilis,  tabes  and  general  paresis. 
(3)  The  Wassermann  test,  using  the  cerebrospinal  fluid  instead  of  blood-serum. 

T.  pertenue. — An  organism  of  similar  morphology  was  first  reported 
by  Castellani  as  present  in  yaws.  It  is  found  in  smears  and  sections  as 
with  T.  pallidum. 

A  point  of  distinction  between  these  spirochaetes  is  that  the  T.  pallidum  is  found 
in  abundance  in  sections  from  a  chancre  about  the  thickened  arteries  in  the  corium, 
while  in  sections  from  a  yaws  nodule  the  T.  pertenue  is  found  chiefly  in  the  region 
of  the  interpapillary  pegs  of  the  Malpighian  layer  of  the  epidermis  where  they  bound 
the  papillary  layer  of  the  corium. 

T.  pertenue  has  been  cultivated  in  the  same  way  as  T.  pallidum  and  Nichols  has 
infected  rabbits  by  intratesticular  injection.  A  disease  of  Guam  known  as  gangosa 
is  possibly  connected  with  a  tertiary  form  of  yaws.  In  persons  who  have  had  yaws 
a  positive  Wassermann  reaction  seems  to  be  given  in  a  higher  percentage  than  is 
true  for  syphilis.  Salvarsan  is  also  more  specific  for  yaws  than  for  syphilis. 

Trypanosoma. 

The  genus  Trypanosoma  has  a  more  or  less  spindle-shaped  body, 
along  one  border  of  which  runs  an  undulating  membrane.  There  is  one 
flagellum  bordering  the  membrane  and  projecting  like  a  whip  posteriorly. 


SLEEPING    SICKNESS  227 

There  is  a  nucleus  (macronucleus)  and  a  blepharoplast  (micronucleus — • 
centrosome),  the  latter  being  located  anteriorly  as  a  chromatin  staining 
dot  or  rod.  From  this  blepharoplast  the  flagellum  proceeds  posteriorly 
bordering  the  undulating  membrane  and  projecting  freely  beyond  the 
posterior  end.  The  nucleus  is  larger,  nearer  the  posterior  end,  and  does 
not  stain  so  intensely  as  the  blepharoplast. 

Some  consider  that  the  trypanosome  developed  from  types  with  a  single  anterior 
flagellum  proceeding  from  a  blepharoplast.  The  moving  of  the  blepharoplast  with 
the  flagellum  to  the  other  end  would  make  the  flagellar  end  the  anterior  end.  This 
controversy  as  to  which  is  the  anterior  end  is  the  cause  of  confusion. 

T.  gambiense. — This  is  the  trypanosome  causing  human  trypano- 
somiasis,  the  latter  stage  of  which  is  known  as  sleeping  sickness.  It  is 
from  17  to  2%/J.  long,  and  from  1.5  to  2/*  wide.  Blepharoplast  oval. 

It  was  first  discovered  in  smears  from  blood  by  Ford  in  1901,  and  recognized  as 
a  trypanosome  by  Dutton  in  1902,  and  observed  in  1903  by  Castellani  in  the  cere- 
brospinal  fluid  of  patients  with  sleeping  sickness.  It  is  now  proposed  to  consider 
cases  where  trypanosomes  are  not  present  in  the  cerebrospinal  fluid  as  in  the  first 
stage;  when  present,  as  in  the  second  stage. 

It  is  very  difficult  to  distinguish  the  human  trypanosome  from  some  of  the  other 
pathogenic  ones  by  staining  methods.  The  immunity  test  is  the  most  reliable. 
An  animal  recovered  from  an  infection  by  a  certain  trypanosome  does  not  possess 
immunity  for  other  pathogenic  ones.  Novy  and  McNeal  cultivated  T.  lewisi  in 
water  of  condensation  on  blood  agar  at  room  temperature  and  Thomson  and  Sinton 
have  recently  cultivated  both  T.  gambiense  and  T.  rhodesiense  by  using  rat's  blood 
instead  of  rabbit's  blood  in  the  N.N.N.  medium.  It  is  present  in  the  blood,  usually 
in  exceedingly  small  numbers,  and  in  the  lymphatic  glands  of  patients.  It  is  by 
puncture  of  the  glands  that  we  have  the  best  means  of  finding  the  parasites.  It  is 
also  found  in  the  cerebrospinal  fluid  in  sleeping  sickness.  The  parasite  stains  readily 
with  Wright's  stain.  The  transmitting  agent  is  the  Glossina  palpalis. 

The  life  history  of  T.  gambiense  is  not  so  well  understood  as  that  of  certain  other 
organisms.  There  seem  to  be  certain  periods  when  even  with  trypanosomes  in  the 
peripheral  circulation  tsetse  flies  do  not  become  infected.  From  about  2  to  5%  of 
flies  seem  to  become  infective  in  experiments.  When  blood  containing  the  so-called 
short  form  of  trypanosomes  is  ingested  by  G.  palpalis  they  reach  the  gut  and  remain 
there  unattached.  From  the  fifth  to  the  seventh  day  they  seem  to  become  scarce 
in  the  digestive  tract  but  later  they  reappear  in  quantity.  About  the  eighth  to  the 
eighteenth  day  long  slender  forms  pass  into  the  proventriculus  and  later  reach  the 
salivary  glands  as  long  slender  forms.  They  multiply  in  the  glands  and  develop  into 
short  crithidial  forms  which  later  become  similar  to  those  found  in  the  peripheral 
circulation.  Robertson  considers  that  the  important  development  takes  place  in 
the  salivary  glands  and  not  in  the  intestine  while  Kleine  thinks  the  mature  forms 
the  first  to  appear  in  the  gut.  It  requires  eighteen  to  twenty  days  or  longer  for  the 
complete  development  and  flies  so  infective  remain  so  for  the  remainder  of  life. 


228  THE   PROTOZOA 

Some  authors  consider  types  representing  male,  female  and  indifferent  forms  to  be 
noted  during  the  developmental  cycle. 

Other  authorities  think  it  possible  that  trypanosomes  may  encyst  in  the  digestive 
tract,  and  so  the  flies  transmit  the  disease  along  with  their  faeces.  This  does  not 
seem  to  be  possible  in  connection  with  human  infections.  Koch  found  several  cases 
where  infection  had  taken  place  by  coitus.  This  is  the  method  of  infection  in  T. 
equiperdum,  a  trypanosome  disease  of  horses. 

The  various  trypanocidal  remedies,  atoxyl,  arsacetine,  etc. ,  have  not  proven  very 
satisfactory.  One  of  Ehrlich's  latest  products,  arsenophenyl-glycine,  however,  has 
given  encouraging  results;  horses  affected  with  surra  having  been  cured  by  its  use. 
In  man  it  has  been  given  in  doses  of  i  gram  without  ill  effects. 

T.  rhodesiense. — -This  is  a  trypanosome  reported  for  man  by 
Stephens  and  Fantham.  The  nucleus,  instead  of  being  in  the  center 
as  in  T.  gambiense,  is  quite  near  the  blepharoplast.  It  is  much  more 
virulent  for  laboratory  animals  than  T.  gambiense.  It  is  transmitted 
by  G.  morsitans  and  the  developmental  cycle  is  similar  to  that  of  T. 
gambiense  except  that  it  seems  that  the  important  developmental  cycle 
occurs  in  the  gut  of  the  fly. 

Schizotrypanum  cruzi  (Trypanosoma  cruzi)  Chagas,  1909.— A 
human  trypanosomiasis  found  in  the  state  of  Minas  Geraes,  in  Brazil,  is 
caused  by  this  protozoon.  Cruz  states  that  the  specific  protozoon  is 
transmitted  by  a  bug  of  the  genus  Conorhinus  (Reduviidae). 

This  trypanosome  is  remarkable  for  the  large  size  of  its  blepharoplast.  In  length 
it  is  only  a  little  longer  than  the  diameter  of  a  red  cell.  It  is  cultivable  on  blood 
agar  and  can  be  transmitted  to  various  laboratory  animals,  as  guinea-pig,  white 
mice,  and  monkeys. 

Cruz  thinks  that  a  non-sexual  cycle  occurs  in  general  tissues  of  man  but  that  a 
special  sexual  cycle  occurs  in  the  lung  capillaries.  In  the  lungs  the  parasite  loses  its 
flagellum  and  becomes  oval  in  shape.  Subsequently  eight  daughter  spores  develop. 
These  spores  or  merozoites  are  liberated  into  the  general  circulation  and  each  one 
penetrates  a  red  cell  and  develops  into  an  adult  trypanosome.  When  ingested  by 
Conorhinus  they  lose  the  flagellum  and  assume  an  oval  Leishmania  form,  which 
multiply  by  fission.  Eventually  there  are  produced  trypanosome  types  which  get 
into  the  salivary  glands  and  thence  into  man.  Chiefly  a  disease  of  children  with 
swelling  of  neck,  axillary  and  groin  glands,  anaemia,  enlarged  spleen,  oedema  of 
eyelids  and  irregular  fever.  Usually  fatal  in  children  but  less  so  in  adult.  In  adults 
apt  to  have  goiter. 

Of  the  more  important  trypanosome  diseases  of  animals  may  be  mentioned: 

1.  Nagana.     Pathogenic  for  domesticated  animals  in  South  Africa.     T.  brucei. 

2.  Surra.     Pathogenic  for  horses  in  India  and  Philippines.     T.  evansi. 

3.  Dourine.     Transmitted  by  coitus  in  horses.     T.  equiperdum. 

4.  Mai  de  caderas.     Affects  horses  in  South  America.     T.  equinum. 

A  harmless  infection,  especially  in  sewer  rats,  is  due  to  T.  lewisi.     Transmission  of 


KALA   AZAR.  22Q 

this  rat  trypanosomiasis  can  apparently  be  brought  about  through  the  agency  of 
both  fleas  and  lice.  In  the  flea  there  is  apparently  a  developmental  cycle  of  a  dura- 
tion of  one  week. 

There  are  many  trypanosomes  in  birds,  fish,  frogs,  etc. 

Trypanoplasma. 

The  genus  Trypanoplasma  has  a  rather  large  blepharoplast,  from  which  arise 
two  flagella.  One  extends  forward  as  a  free  anterior  flagellum,  while  the  other 
projects  posteriorly,  running  along  the  border  of  the  undulating  membrane.  This 
genus  is  not  known  for  man. 

Leishmania. 

The  genus  Leishmania  includes  three  species:  L.  donovani,  the  para- 
site of  kala  azar,  L.  tropica,the  parasite  of  oriental  sore  andL.  infantum 
the  cause  of  a  leishmaniasis  among  children  in  northern  Africa. 

The  disease  known  as  ponos,  which  exists  in  the  Grecian  islands  Spezzia  and 
Hydra  has  been  found  by  Galle  to  be  a  leishmaniasis.  Nicolle  has  found  a  disease 
of  very  young  children  (as  a  rule  in  the  second  year  of  life)  in  Tunis  due  to  L. 
infantum.  This  protozoon  morphologically  resembles  L.  tropica  but  is  smaller. 
It  is  found  chiefly  in  the  spleen,  liver  and  bone  marrow.  The  symptoms  are  extreme 
anaemia,  splenic,  and  to  a  less  degree,  hepatic  enlargement.  Irregular  temperature, 
rapid  pulse  and  a  mononuclear  leukocytosis  and  transient  oedema  are  also  noted. 
It  can  be  inoculated  into  the  dog  and  monkey;  other  animals  are  practically  immune. 

A  similar  disease  has  been  noted  in  Italy,  Malta,  and  Portugal.  L.  infantum 
grows  rapidly  in  Novy  MacNeal  medium,  in  which  medium  L.  donovani  will  not  grow. 
Furthermore  inoculation  of  L.  donovani  into  dogs  and  monkeys  has  been  unsuccess- 
ful. These  are  undoubtedly  different  species,  inasmuch  as  in  sections  of  India, 
where  tropical  ulcer  was  common,  there  was  no  kala  azar,  and  in  Assam  where  kala 
azar  prevailed  there  were  no  Leishman-Donovan  bodies  to  be  found  in  smears  from 
the  tropical  ulcerations  there  present,  except  rarely  in  cases  of  general  infection. 
L.  tropica  has  been  cultivated  by  Nicolle. 

It  is  interesting  that  the  parasite  of  kala  azar  cannot  be  cultivated  except  in 
sterile  media  while  that  of  oriental  sore  will  grow  in  media  contaminated  with  cocci. 

These  parasites  are  typically  intracellular,  being  within  either  polymorpho- 
nuclears,  which  contain  only  one  or  two  of  the  bodies,  or  in  large  mononuclears,  in 
which  there  may  be  as  many  as  six.  They  may  be  packed,  however,  in  phagocytic 
endothelial  cells. 

In  kala  azar  smears  taken  during  life  we  may  find  the  bodies  imbedded  in  a  faintly 
blue  staining  matrix;  after  death  and  in  sections  of  tissue  such  an  appearance  is 
not  seen.  In  the  spleen  they  are  not  found  in  the  Malpighian  bodies,  but  in  the 
phagocytic  cells  lining  the  lymph  spaces.  The  parasites  occur  in  the  peripheral 
circulation  in  about  80%  of  the  cases.  They  abound  in  the  liver  and  spleen.  The 
parasite  is  oval  and  about  2  X  3/£.  There  are  two  distinct  chromatin  staining  masses. 
The  larger  nucleus  is  more  or  less  spherical,  peripherally  situated,  and  stains  faintly, 


230  THE    PROTOZOA 

while  the  smaller  chromatin  mass  is  generally  rod-shaped  and  stains  intensely.  It 
has  been  recently  recommended  that  instead  of  liver  or  splenic  puncture  for  the 
demonstration  of  these  bodies,  a  blister  be  raised  and  a  smear  from  that  containing 
many  polymorphonuclears  might  show  these  bodies.  The  affection  is  characterized 
by  a  leukopaenia  so  that  it  is  very  difficult  to  demonstrate  the  parasites  in  ordinary 
blood  smears. 

By  cultivating  the  parasites  obtained  from  splenic  puncture  in  acidified  sodium 
citrate  solution  at  room  temperature,  Rogers  succeeded  in  obtaining  flagellated  forms 
similar  to  Herpetomonas.  An  anterior  flagellum  proceeds  directly  from  the  blephar- 
oplast.  The  bedbug  is  supposed  to  be  the  intermediary  host. 

Patton  has  recently  noted  that  when  bedbugs  feed  on  kala-azar  patients  who  have 
the  L.  D.  bodies  in  their  peripheral  circulation  that  the  parasites  develop  into  the 
flagellate  stage  in  the  bedbug  and  are  present  in  great  numbers  from  the  fifth  to  the 
eighth  day.  These  flagellate  forms  change  into  postflagellate  ones  by  the  twelfth 
day  and  are  then  found  in  the  stomach.  If,  however,  he  allowed  the  bedbugs  to 
have  a  second  feeding  of  human  blood  after  the  infecting  feeding  the  flagellates  dis- 
appeared within  twelve  hours.  This  is  apparently  an  important  point  in  epidemi- 
ology. Patton  succeeded  in  infecting  a  white  rat  with  intraperitoneal  injection  of 
splenic  emulsion  from  a  kala-azar  patient. 

L.  infantum  is  transmitted  from  dog  to  dog  by  the  dog  flea,  P.  serraticeps  and 
the  same  agent  probably  transfers  the  parasite  from  dog  to  man.  Experiments 
would  indicate  that  the  Indian  form  of  kala  azar  is  not  a  disease  which  can  be  trans- 
mitted to  dogs. 

The  genera  Herpetomonas  and  Crithidia  are  frequently  found  in  the  alimentary 
tract  of  insects  and  have  caused  confusion  in  the  search  for  developmental  forms  of 
various  pathogenic  flagellates  in  transmitting  insects.  In  Herpetomonas,  of  which 
the  type  species  is  H.  muscae  domesticae,  the  body  is  spindle-shaped  with  a  rather 
blunt  flagellar  end  and  an  attenuated  anterior  end.  In  Crithidia  both  extremities 
are  pointed  and  the  blepharoplast  is  situated  toward  the  center  quite  near  the  tropho- 
nucleus.  In  Herpetomonas  the  blepharoplast  is  near  the  rather  blunt  flagellar 
extremity  at  some  distance  from  the  nucleus. 

There  is  no  undulating  membrane  in  either  of  these  genera,  this  differentiating 
them  from  Trypanosoma. 

Darling  has  reported  from  Panama  a  protozoon  somewhat  like  Leishmania  in 
which  the  cells  of  lungs,  liver,  spleen,  and  lymphatic  glands  contained  numerous 
parasites  about  3  to  4/4  in  diameter,  slightly  oval  in  outline,  and  containing  a  large 
and  small  chromatin  staining  mass.  He  has  given  it  the  name  Histoplasma 
capsulata. 


Trichomonas. 

Trichomonas  vaginalis. — This  parasite  has  a  fusiform  body  and  is  about 
It  has  three  flagella  arising  from  the  anterior  end  and  an  undulating  membrane.  It 
lives  in  vaginal  mucus  which  has  an  acid  reaction  A  change  of  reaction,  as  at  men- 
struation, causes  them  to  disappear.  Forms  similar  to  the  T.  vaginalis  have  been 
found  in  the  intestine  and  in  sputum  from  putrid  bronchitis. 


INFUSORIA.  231 

These  flagellates  are  generally  considered  harmless,  although  doubt  as  to  this  is 
expressed  by  some  authors. 

Lamblia. 

Lamblia  intestinalis. — These  parasites  are  aboutioX  i5/*  and  have  a  pear-shaped 
body  with  a  depression  at  the  blunt  anterior  end.  This  depression  enables  the 
flagellate  to  attach  itself  to  the  summit  of  an  epithelial  cell.  Around  the  depression 
are  three  pairs  of  flagella  which  are  constantly  in  motion.  Another  pair  of  flagella 
project  from  either  side  of  the  blunt  little  tail-like  projection.  When  stained,  the 
parasites  have  a  pyriform  shape  with  two  chromatin  staining  areas  on  either  side 
of  the  anterior  end.  When  encysted,  they  assume  an  oval  shape.  This  parasite 
is  generally  considered  as  of  little  importance,  but  inasmuch  as,  when  in  great  num- 
bers in  the  caecum  and  appendix,  they  may  give  rise  to  symptoms  resembling  appen- 
dicitis and  as  they  are  responsible  for  a  chronic  and  intractable  diarrhoea  associated 
with  mental  and  physical  depression,  this  is  undoubtedly  an  affection  only  minor  in 
importance  to  amoebic  infection.  It  is  a  common  infection  in  the  tropics. 

INFUSORIA  (CILIATA). 

The  Infusoria  are  the  most  highly  developed  of  the  Protozoa. 

The  bodies  of  Infusoria  are  oval  and  may  be  free  or  attached  to  a  stalk-like 
contractile  pedicle,  as  with  Vorticella,  or  they  may  be  sessile.  The  cilia,  which  are 
characteristic,  may  be  markedly  developed  around  the  cytostome  (mouth)  and  serve 
the  purpose  of  directing  food  into  the  interior,  while  others  act  as  locomotor  organs. 
The  body  is  enveloped  by  a  cuticle  which  may  only  have  one  opening  or  slit,  to  serve 
as  mouth;  or  it  may  have  a  second  one,  a  cytopyge  or  anus.  Usually  the  faecal 
matter  is  ejected  through  a  pore  which  may  be  visible  only  when  in  use.  They  usu- 
ally have  a  large  nucleus  and  a  small  one.  Infusoria  tend  to  encyst  when  conditions 
are  unfavorable  (as  when  water  dries  up  in  a  pond).  When  the  cilia  are  evenly 
distributed  over  the  entire  body  of  the  ciliates  we  have  the  order  Holotricha;  when 
ciliated  all  over,  but  with  more  prominent  cilia  surrounding  the  peristome,  we  call 
the  order  Heterotricha.  It  is  to  this  order  that  the  Infusoria  of  man  belong. 

Balantidium  coli. — -This  is  the  only  ciliate  of  importance  in  man. 
It  is  a  common  parasite  of  hogs.  It  is  from  60  to  ioo/i  long  by  50  to 
'jofJ.  broad,  and  has  a  peristome  at  its  anterior  end  which  becomes  narrow 
as  it  passes  backward.  It  has  an  anus.  The  ectosarc  and  the  endosarc 
are  distinctly  marked.  The  cuticle  is  longitudinally  striated. 

These  parasites  cause  an  affection  similar  to  dysentery  and  may  bring  about  a 
fatal  termination.  It  is  almost  impossible  to  escape  noticing  the  actively  moving 
bodies  if  a  faecal  examination  is  made.  When  encysted  they  are  round. 

Another  ciliate,  the  B.  minimum,  25  x  15^,  has  also  been  reported  for  man. 

Nyctotherus  faba  has  a  kidney-shaped  body  and  is  about  25  by  15;*.  It  has  a 
large  contractile  vacuole  at  the  posterior  end.  It  has  a  large  nucleus  in  the  center 
with  a  small  fusiform  micronucleus  lying  close  to  it.  It  has  only  been  reported 
once  for  man. 


DESCRIPTION  OF  PLATE  I. 

(Kolle  and  Wassermann. ) 

Malarial  Parasites. 

4 

1.  Two  tertian  parasites  about  thirty-six  hours  old,  attacked  blood-corpuscles 
swollen. 

2.  Tertian  parasite  about  thirty-six  hours  old ;  stained  by  Romanowsky's  method. 
The  black  granule  in  the  parasite  is  not  pigment  but  chromatin.     Next  to  it  and  to 
the  left  is  a  large  lymphocyte,  and  under  it  the  black  spot  is  a  blood  plate. 

3.  Tertian  parasite,  division  form  nearby  is  a  polynuclear  leukocyte. 

4.  Quartan  parasite,  ribbon  form. 

5.  Quartan  parasite,  undergoing  division. 

6    Tropical  fever  parasite,     (^stivo-autumnal.)     In  one  blood-corpuscle  may 
b2  seen  a  smaller,  medium,  and  large  tropical  fever-ring  parasite. 

7.  Tropical  fever  parasite.     Gametes  half-moon  spherical  form.     Smear  from 
bone  marrow. 

8.  Tropical  fever  parasite  which  is  preparing  for  division  heaped  up  in  the  blood 
capillaries  of  the  brain. 

Asexual  Forms. 

9.  Smaller  tertian  ring  about  twelve  hours  old. 

10.  Tertian  parasite  about  thirty-six  hours  old,  so-called  amoeboid  form. 

1 1.  Tertian  parasite  still  showing  ring  form  forty-two  hours  old. 

12:  Tertian  parasite,  two  hours  before  febrile  attack.     The  pigment  is  beginning 
to  arrange  itself  in  streaks  or  lines. 

13.  Tertian  parasite  further  advanced  in  division.     Pigment  collected  in  large 
quantities. 

14.  Further  advanced  in  the  division.     (Tertian  parasite.) 


232 


PLATE  I. 


COCCIDIA.  233 

SPOROZOA. 

This  class  of  Protozoa  gets  its  name  from  the  method  of  reproduction 
— sporulation.  These  parasites  rarely  show  binary  fission.  While 
the  sporozoa  are  found  within  cells,  in  the  tissues  and  in  internal  cav- 
ities, as  intestine  and  bile  ducts,  yet  it  is  as  inhabitants  of  the  blood  that 
they  have  their  greatest  importance  for  man — -these  are  known  as 
Haemosporidia.  A  sporozoon  may  be  either  naked  or  amceboid  or  be 
covered  with  a  distinct  cuticle. 

NOTE. — Sporozoa  are  divided  into  two  subclasses — the  Telosporidia  and  the  Neo- 
sporidia.  In  the  former  the  vegetative  activity  of  the  protozoon  goes  on  to  full  growth 
at  which  time  the  reproductive  activity  commences.  With  the  Neosporidia, 
however,  the  growth  and  reproduction  go  on  at  the  same  time. 

Among  the  Telosporidia  we  have  the  orders  Gregarinaria,  Coccidiaria,  and 
Haemosporidia 

Gregarines  are  chiefly  parasites  of  arthropods  and  worms  and  are  not  known 
for  man  or  the  higher  vertebrates. 

The  subclass  Neosporidia  is  practically  of  no  importance  in  human  parasitology, 
only  the  order  Sarcosporidia  having  been  reported  for  man.  From  an  economic 
standpoint,  however,  the  order  Myxosporidia  is  of  great  importance — Nosema  being 
the  cause  of  pebrine,  a  disease  destructive  to  the  silkworm.  In  this  the  eggs  of  an 
infected  N.  bombycis  may  be  infected. 

Coccidiaria. 

The  parasites  of  the  order  Coccidiaria  are  almost  exclusively  found  in  the  intes- 
tines and  in  the  organs  connected  with  it.  In  the  vegetative  stage  it  lives  within  an 
epithelial  cell,  which  it  destroys.  Afterward  it  falls  into  the  lumen  lined  by  this 
epithelial  cell  and  sporulates,  either  by  the  method  of  schizogony  or  sporogony. 

Owing  to  their  egg-like  shape,  coccidia  have  often  been  considered  as  the  ova  of 
intestinal  parasites,  and  vice  versa.  Upon  swallowing  an  oocyst  with  its  contained 
sporozoites  the  membrane  of  the  oocyst  is  digested  in  the  duodenum  and  the  sporo- 
zoites  liberated.  They  enter  epithelial  cells,  as  of  intestine,  and  reproduce  by 
schizogony.  After  a  varying  number  of  nonsexual  cycles  sporogony  commences, 
sporonts  being  produced  instead  of  schizonts.  The  female  sporont  is  fertilized  by 
the  microgamete  which  is  an  elongated  body  provided  with  two  flagella.  These 
microgametes  are  formed  from  the  male  sporont  and  when  thrown  off  from  the 
periphery  they  enter  (usually  a  single  one)  the  macrogamete.  After  fertilization  a 
resistant  membrane  is  formed  and  the  term  oocyst  is  used.  Within  the  oocyst 
are  found  smaller  cysts,  the  sporocysts,  in  which  the  sporozoites  are  formed. 

The  cycle  is  very  similar  to  that  of  malaria  except  that  no  arthropod  host  is 
required  for  the  sexual  cycle.  The  spores  which  are  formed  in  schizogony  are  known 
as  merozoites. 

Merozoites  may  best  be  distinguished  from  sporozoites  by  the  presence  of  a 
nuclear  karyosome,  this  being  absent  in  sporozoites.  In  Eimeria  we  have  the 
oocyst  containing  four  sporocysts  with  two  sporozoites  in  each  sporocyst  while  in 
Isospora  we  have  an  oocyst  containing  two  sporocysts  with  four  sporozoites  in  each. 

Eimeria  stiedae. — This  sporozoon  is  usually  known  as  the  Coccidium  cuniculi 


DESCRIPTION  OF  PLATE  II. 

(Kolle  and  Wassermann. ) 

Malarial  Parasites. 

15.  Complete  division  of  the  parasite.     Typical  mulberry  form. 

1 6.  To  the  left  is  the  completed  division  form,  an  almost  developed  gamete,  which 
is  to  be  recognized  by  its  dispersed  pigment. 

1 7.  A  tertian  ring  parasite,  small  size  broken  up. 

1 8.  Three-fold  infection  with  tertian  parasite.     The  oval  black  granules  are  the 
chromatin  granules. 

19.  To  the  left,  tertian  parasite  with  large,  sharply  demarked,  and  deeply  colored 
chromatin  granules.     To  the  right,  tertian  parasite.     Both  thirty-six  hours  old. 
Both  probably  gametes. 

20.  Tertian  parasite  thirty-six  hours  old,  ring  form. 

21.  Tertian  parasite  with  beginning  chromatin  division,  with  eight  chromatin 
segments. 

22.  Tertian  parasite  chromatin  division  farther  advanced  with  twelve  chromatin 
granules,  in  part  triangular  in  form. 

23.  Completed  division  figure  of  a  tertian  parasite.     Twenty-two  chromatin 
granules. 

24.  The  young  tertian  parasites  separating  themselves  from  each  other.     The 
pigment  remains  behind  in  the  middle. 

25.  Quartan  ring  parasite,  which  is  hard  to  differentiate  from  large  tropical  ring 
or  small  tertian  ring. 

26.  Quartan  ring  lengthening  itself. 

27.  Small  quartan  ribbon  form. 

28.  The  quartan  ribbon  increases  in  width.     The  dark  places  consist  almost 
entirely  of  pigment. 


234 


PLATE  II. 


MALARIA  235 

or  C.  oviforme.  It  is  most  frequently  found  in  the  epithelium  of  the  bile  ducts. 
It  has  very  rarely  been  reported  for  man.  In  these  cases  (about  five)  cysts  of  the 
liver  have  been  found  containing  coccidia.  The  parasite  is  about  40  X  20^,  and  is 
oval  in  shape  with  a  double  outlined  integument.  The  sporozoites,  which  form 
inside,  are  falciform  in  shape.  These  escape  and  enter  fresh  epithelial  cells,  and 
thus  the  process  of  schizogony  goes  on.  The  parasites  of  the  liver  are  larger  than 
those  found  in  the  intestines,  these  latter  being  only  about  3oX  is/*.  In  the  faeces 
the  form  most  often  found  is  the  oocyst,  about  40  X  2O//.  Infection  takes  place  by 
ingestion  of  the  oocyst. 

Isospora  bigemina. — This  parasite,  formerly  called  the  Coccidium  bigeminum, 
lives  in  the  intestinal  villi  of  dogs  and  cats.  It  is  about  12  X  8ft.  and  shows  a  highly 
refractile  envelope  (oocyst)  containing  two  biscuit-shaped  sporocysts  within  each  of 
which  are  four  sporozoites.  It  has  been  reported  for  man  three  times. 

Haemosporidia. 

Of  the  Sporozoa  found  in  the  blood  (Haemosporidia) ,  the  malarial 
parasites  are  the  only  ones  connected  with  disease  in  man. 

In  addition  to  man,  infections  with  parasites  of  a  similar  nature  are  found  in 
monkeys  (Plasmodium  kochi;  the  sexual  forms  alone  seem  to  be  present),  in  birds 
(Hsemamceba  relicta;  this  organism  is  usually  designated  Proteosoma) .  An  infection 
of  crows  and  pigeons  of  like  nature  is  Halteridium.  Numerous  haemosporidia 
have  been  reported  for  bats,  various  other  mammals,  tortoises,  lizards,  etc. 

The  life  history  of  the  malarial  parasite  is  one  of  the  most  interesting  chapters 
in  medicine.  Laveran  discovered  the  parasite  in  1880.  In  1885,  Golgi  noted  that 
sporulation  occurred  simultaneously  at  time  of  malarial  paroxysm.  Koch,  Golgi, 
and  Celli  demonstrated  existence  of  different  species  for  different  types  of  fever. 
King  and  Laveran  (1884)  considered  possibility  of  mosquito  transmission.  Manson 
(1894)  formulated  hypothesis  that  gametes  were  destined  to  undergo  development 
in  the  mosquito  from  observing  that  flagellated  bodies  only  appeared  some  time 
after  the  blood  was  withdrawn. 

Ross  (1895)  demonstrated  that  flagellation  takes  place  in  the  stomach  of  the 
mosquito.  McCallum  (1897)  saw  fertilization  of  macrogametes  by  microgametes 
of  Halteridium.  Opie  recognized  differences  in  sexual  characteristics. 

Ross  (1898)  demonstrated  life  cycle  of  bird  malaria  (Proteosoma),  showing  for- 
mation of  zygotes  and  presence  of  sporozoites  in  salivary  glands.  Grassi  and  Big- 
nami  proved  the  cycle  for  Anophelinae  for  human  malaria.  In  1900  (Sambon  and 
Low),  infected  mosquitoes  from  Italy  were  sent  to  London,  where,  by  biting,  they 
infected  two  persons. 

Life  History. — When  man  is  at  first  infected  by  sporozoites  we  have 
starting  up  a  nonsexual  cycle  which  is  completed  in  from  forty-eight  to 
seventy-two  hours,  according  to  the  species  of  parasite.  The  falciform 
sporozoite  bores  into  a  red  cell,  assumes  a  round  shape  and  continues 
to  enlarge  (schizont).  Approaching  maturity,  it  shows  division  into  a 
varying  number  of  spore-like  bodies.  At  this  stage  the  parasite  is 


DESCRIPTION  OF  PLATE  III. 

(Kolle  and  Wassermann.) 

Malarial  Parasite. 

29>  3°>  31-  The  quartan  ribbon  increases  in  width.     The  dark  places  consist 
almost  entirely  of  pigment. 

32.  Beginning  division  of  the  quartan  parasite  and  the  black  spot  in  the  middle 
is  the  collected  pigment. 

33.  Quartan  ring. 

34.  Double  infection  with  quartan  parasites. 

35.  Wide  quartan  band.     The  fine  black  stippling  in  the  upper  half  of  the  para- 
site is  pigment. 

36.  Beginning  division  of  the  quartan  parasite.     The  chromatin  (black  fleck) 
is  split  into  four  parts. 

37.  Division  advanced,  quartan  parasites. 

38.  Typical  division  figure  of  the  quartan  parasite. 

39.  Finished  division  of  the  quartan  parasite.     Ten  young  parasites,  pigment  in 
the  middle. 

40.  Young  parasites  separated  from   one  another. 

41.  Small  and  medium  tropical  ring,  the  latter  in  a  transition  stage  to  a  large 
tropical  ring. 

42.  Small,  medium  and  large  tropical  ring,  together  in  one  corpuscle. 


236 


PLATE  III. 


16 


MALARIA 


237 


termed  a  merocyte.     When  the  merocyte  ruptures,  these  spore-like 
bodies  or  merozoites  enter  a  fresh  cell  and  develop  as  before. 

At  the  time  that  the  merocyte  ruptures  it  is  supposed  that  a  toxin  is 
given  off  which  causes  the  malarial  paroxysm.  The  cycle  goes  on  by 
geometric  progression  from  the  first  introduction  of  the  sporozoite,  but 
it  is  usually  about  two  weeks  before  a  sufficient  number  of  merocytes 
rupture  simultaneously  to  produce  sufficient  toxin  for  symptoms  (period 
of  incubation).  This  cycle  is  termed  schizogony. 


9. 


FIG.  62. — Sexual  and  nonsexual  cycle  of  malaria,  i,  Schizonts;  2,  merocyte; 
3,  merozoites;  4,  macrogamete;  5,  microgametocyte;  6,  and  7,  gametes  in  stomach  of 
mosquito;  8,  microgametocyte  throwing  off  microgametes;  9,  microgamete  fertilizing 
macrogamete;  10,  vermiculus  or  zygote;  n  and  12,  zygotes;  13,  zygote  distended 
with  sporozoites;  14,  sporozoites. 

After  a  varying  time,  whether  by  reason  of  necessity  for  renewal  of 
vigor  of  the  parasite  by  a  respite  from  sporulation,  or  whether  from  a 
standpoint  of  survival  of  the  species,  sexual  forms  (gametes)  develop. 
Some  think  that  sporozoites  of  sexual  and  nonsexual  characteristics  are 
injected  at  the  same  time.  It  is  usually  considered,  however,  that 
sexual  forms  develop  from  pre-existing  nonsexual  parasites. 

These  gametes  show  two  types:  the  one  which  contains  more  pig- 
ment, has  less  chromatin,  and  stains  more  deeply  blue  is  the  female — -a 
macrogamete;  the  other  with  more  chromatin,  less  pigment,  and  staining 


DESCRIPTION  OF  PLATE  IV. 

(Kolle  and  Wassermann. ) 

Malarial  Parasite. 

43.  To  the  left  a  young  (spore)  tropical  parasite.     To  the  right  a  medium  and 
large  tropical  parasite. 

44.  An  almost  fully  developed  tropical  parasite.     The  black  granules  are  pig- 
ment heaps. 

45.  Young  parasites  separated  from  one  another.     Broken  up  division  forms 
twenty-one  new  parasites. 

46.  To  the  left  a  red  blood-corpuscle  with  basophilic,  karyochromatophilic  gran- 
ules.    Prototype  of  malarial  parasite.     On  the  right  a  red  blood-corpuscle  with  re- 
mains of  nucleus. 

Sexual  Forms  or  Gametes. 

47.  An  earlier  quartan  gamete  (macrogamete  in  sphere  form),  female. 

48.  An  earlier  quartan  gamete  (microgametocyte),  male. 

49.  Tertian  gamete,  male  form  (microgametocyte). 

50.  Tertian  gamete,  female  (macrogamete). 

51.  Tertian  gamete  (microgametocyte)  still  within  a  red  blood-corpuscle. 

52.  Macrogamete  tertian  within  a  red  blood-corpuscle. 

53.  Tropical    fever.     (^Estivo-autumnal)    gamete,    half-moon     (crescent)    still 
lying  in  a  red  blood-corpuscle.     In  the  middle  is  the  pigment.     The  concave  side 
of  the  crescent  is  spanned  by  the  border  of  the  red  blood-corpuscle. 

54.  Gamete,  tropical  fever  parasite. 

55.  Gamete  of  tropical  fever  parasite  heavily  pigmented. 

56.  Gamete  of  the  tropical  fever  parasite  (flagellated  form),  microgametocyte 
sending  out  microgametes  (flagella  or  spermatozoa). 


238 


PLATE  IV. 


MALARIA  239 

grayish-green  rather  than  blue  is  the  male— a  microgametocyte.  When 
the  gametes  are  taken  into  the  stomach  of  the  Anophelinae,  the  male  cell 
throws  off  spermatozoa-like  projections,  which  have  an  active  lashing 
movement  and  break  off  from  the  now  useless  cell  carrier  and  are  there- 
after termed  microgametes.  These  fertilize  the  macrogametes  and  this 
body  now  becomes  a  zygote. 

By  a  boring-like  movement  the  zygote  goes  through  the  walls  of  the 
mosquito's  stomach,  stopping  just  under  the  outer  epithelial  layer  of 
the  stomach  or  mid-gut.  It  continues  to  enlarge  until  about  the  end 
of  one  week  it  has  grown  to  be  about  6o/*  in  diameter  and  has  become 
packed  with  hundieds  of  delicate  falciform  bodies. 

Zygotes  of  benign  tertian  show  little  rod-like  particles  of  yellowish  pigment — 
those  of  malignant  tertian  black  clumps,  which,  however,  are  not  so  coarse  as  those 
of  quartan. 

The  matuie  zygote  now  ruptures  and  the  sporozoites  are  thrown  off 
into  the  body  cavity.  They  make  their  way  to  the  salivary  glands  and 
thence,  by  way  of  the  veneno-salivary  duct  in  the  hypopharynx,  they 
are  introduced  into  the  circulation  of  the  person  bitten  by  the  mosquito, 
and  start  a  nonsexual  cycle.  As  the  sexual  life  takes  place  in  the  mos 
quito,  this  insect  is  the  definitive  host —  man  is  only  the  intermediary  host. 

It  must  be  remembered  that  only  certain  genera  and  species  of  Anophelinae  are 
known  malaria  transmitters;  thus  Stephens  and  Christophers,  in  dissecting  496 
mosquitoes  of  the  species  M.  rossi,  did  not-  find  a  single  gland  infected  with  sporo- 
zoites. With  M.  culicifacies,  however,  twelve  in  259  showed  infection. 

This  is  one  of  the  methods  of  determining  the  endemicity  of  malaria  or  the 
malarial  index.  There  are  two  other  methods:  i.  by  noting  the  prevalence  of 
enlarged  spleen,  and  2.  by  determining  the  number  of  inhabitants  showing  malarial 
parasites  in  the  blood.  This  index  is  best  determined  from  children  between  two 
and  ten  years  of  age,  as  children  under  two  years  show  too  high  a  proportion  of  para- 
sites in  the  peripheral  blood  while  those  over  ten  years  of  age  show  too  great  an  in- 
cidence of  enlarged  spleens. 

There  are  three  species  of  malarial  parasites:  i.  the  Plasmodium 
vivax,  that  of  benign  tertian — cycle,  forty-eight  hours;  2.  the  Plasmo- 
dium malariae,  that  of  quartan — cycle,  seventy- two  hours;  and  3.  the 
Plasmodium  falciparum,  that  of  aestivo-autumnal  or  malignant  tertian 
— cycle  of  forty-eight  houis. 

Variations  in  cycles  may  be  produced  by  infected  mosquitoes  biting  on  successive 
nights,  so  that  one  crop  will  mature  and  sporulate  twenty-four  hours  before  the 
second.  This  would  give  a  quotidian  type  of  fever.  In  aestivo-autumnal  infections 
anticipation  and  retardation  in  the  sporulation  cause  a  very  protracted  paroxysm, 
lasting  eighteen  to  thirty-six  hours;  this  tends  to  give  a  continued  or  remittent  fever 
instead  of  the  characteristic  type. 


240 


THE   PROTOZOA 


UNSTAINED  SPECIMEN  (FRESH  BLOOD). 


P.  vivax. 
'(Benign  tertian.) 

P.  malarias. 
(Quartan.) 

P.  falciparum. 
(Malignant  tertian) 
Costive-autumnal.  ) 

Character  of  the 

Swollen  and  light 

About    the    size 

Tendency  to  distortion 

infected  red  cell. 

in   color  after 

and  color  of  a 

of  red  cell  rather  than 

eighteen  hours. 

normal  red  cell. 

crenation.      Shriveled 

appearance.     (Brassy 

color.  ) 

Character  of  young 

Amoeboid  outline. 

Frosted  glass 

Small,  distinctly  round, 

schizont. 

Hyaline.     Rare- 

disc.    Very 

crater-like  dots  not 

ly     more     than     slight  amoeboid 

more  than  one-sixth  di- 

one in  r.c.     Ac- 

motion. 

ameter  of  red  cell. 

tive      amoeboid 

Two  to  four  parasites 

movement. 

in  one  red  cell  common. 

One  third  diam. 

of  r.  c. 

Character  of  ma- 

Amoeboid outline. 

Rather  oval  in 

Only  seen  in  overwhelm- 

ture schizont. 

No        amoeboid 

shape.     Slug- 

ing infection.     Have 

movement. 

gish  movement 

scanty  fine  black  pig- 

of peripherally 

ment  clumped  together. 

placed    coarse 

black  pigment. 

Pigment. 

Fine  yellow 

Coarse      almost 

Pigmented  schizonts 

brown  granules 

black.      Shows 

very  rare  in  periph. 

which  show  ac- 

movement only 

circulation  except  in 

tive   motion  in 

in     young     to 

overwhelming  infec- 

one-half  grown 

half-grown 

tions.     Tend  to  clump 

schizont.     Mo- 

schizont. 

as  excentric  pigment 

tion  ceases  in 

mass  blocks. 

full-grown 

schizont. 

STAINED  SPECIMEN. 


P.  vivax. 
(Benign  tertian.) 

P.  malariae. 
(Quartan.  ) 

P.  falciparum. 
(Malignant  tertian) 
C^Estivo-autumnal.  ) 

Character  of  in- 
fected red  cell. 

Larger  and  light- 
er pink  than 
normal  red  cell. 
Shows  "Schiiff- 
ner's  dots." 

About  normal 
size  and  stain- 
ing. 

Shows  distortion  and 
some  polychromato- 
philia  and  stippling. 
Rarely  we  have  coarse 
cleft-like  reddish  dots 
—  Maurer's  spots. 

MALARIA 


STAINED  SPECIMEN. — (Continued.) 


241 


P.  vivax. 
(Benign  tertian) 

P.  malariae 
(Quartan.) 

P.  falciparum 
(Malignant  tertian) 
(^stivo-autumnal.) 

Character  of  young 

Chromatin  mass 

Rather  thick 

Very  small  sharp  hair- 

schizont. 

usually  single 

round  rings 

like  rings,  with  a 

and  situated  in 

which  soon  tend 

chromatin     mass    pro- 

line with  the 

to  show  as  equa- 

truding from  the  ring. 

ring  of  the  ir- 

torial bands. 

Often  appears  on  per- 

regularly out- 

iphery of  red  cell  as  a 

lined  blue  para- 

curved blue  line  with 

site. 

prominent  chromatin 

dot.     Frequently     two 

chromatin  dots. 

Character  of  half- 

Vacuolated  loop- 

More  marked 

tNot  often  found  in  per- 

grown schizont. 

ed-like  body 

band  forms 

ipheral  circulation. 

with  single  chro- 

stretching 

Chromatin  still  com- 

matin aggrega- 

across r.  b.  c. 

pact. 

tion.     Schiiff- 

ners  dots. 

Character  of  ma- 

Fine pigment 

Coarse  pigment 

Very  rarely  seen  in  per- 

ture schizont. 

rather  evenly 

rather  peripher- 

ipheral circulation  in 

distributed  in 

ally  arranged  in 

ordinary  infection. 

irregularly  out- 

an oval  para- 

Pigment clumps  early. 

lined  parasite. 

site. 

Character  of  me- 

Irregular  division 

Rather  regular        Sporulation  occurs  in 

rocyte. 

into  fifteen  or 

division  into            spleen,  brain,  etc. 

more  spore-like 

eight  or  ten 

Rarely  in  peripheral 

chromatin  dot 

merozoites  — 

circulation.     Eight  to 

segments. 

Daisy. 

ten  chromatin  staining 

merozoites. 

Character  of  mac- 

Round  deep  blue. 

Round,  similar 

Crescentic,  deep  blue, 

rogamete. 

Abundant, 

to  P.  vivax  but 

pigment  clumped  at 

rather  coarse 

smaller. 

center,  chromatin    ' 

pigment,  chro- 

scanty and  in  center. 

matin  at  per- 

iphery. 

Character  of  mi- 

Round,  light 

Round  like  P. 

More  sausage-shaped 

crogametocyte. 

green-blue,  pig- 

vivax. 

than  crescent.     Light 

ment  less  abun- 

blue.    Pigment  scat- 

dant, chroma- 

tered  throughout. 

tin  abundant 

Chromatin  scattered. 

and  located 

centrally  or  in 

a  band. 

242  THE    PROTOZOA 

In  full  grown  schizonts  we  find  the  chromatin  in  separate  aggregations  through- 
out the  parasite  while  the  pigment  is  clumped.  In  gametes  the  pigment  is 
scattered  and  the  chromatin  is  in  a  single  mass. 

If  many  young  ring  forms  are  present  during  pyrexia  it  is  probable  that  the 
infection  is  E.A. 

In  parthenogenesis,  as  observed  in  P.  vivax,  the  nonsexual  forms  and  the  males 
die  off  leaving  only  the  female  forms.  The  nucleus  divides  into  a  dense  and  light 
portion.  The  latter  degenerates  and  the  former  goes  on  to  merozoite  formation. 

This  is  Schaudinn's  explanation  of  relapses.  Another  explanation  of  latent 
malaria  is  by  conjugation  of  two  ring  forms. 

In  the  diagnosis  of  malaria  one  should  always  examine  both  a  fresh 
specimen  and  a  stained  one,  as  each  method  gives  valuable  information 
in  differentiating  species.  When  time  will  not  permit  the  examination 
by  both  methods,  always  use  the  smear  stained  by  Wright's  stain,  as 
the  small  peripherally  situated  rings  of  aestivo-autumnal  fever  may 
escape  notice  in  a  fresh  specimen. 

For  the  cultivation  of  malarial  parasites  (Bass)  the  blood  in  10  to  20  c.c.  quan- 
tities is  taken  from  the  patient's  vein  and  received  in  a  centrifuge  tube  which  con- 
tains i/io  c.c.  of  50%  glucose  solution.  A  glass  rod,  or  piece  of  tubing,  extending 
to  the  bottom  of  the  centrifuge  tube  is  used  to  defibrinate  the  blood.  After  centrif u- 
galizing  there  should  be  at  least  i  inch  of  serum  above  the  cell  sediment.  The 
parasites  develop  in  the  upper  cell  layer  about  1/50  to  1/20  inch  from  the  top.  All 
of  the  parasites  contained  in  deeper  lying  red  cells  die.  To  observe  the  development, 
red  cells  from  this  upper  i/20-inch  portion  are  drawn  up  with  a  capillary  bulb  pipette. 

Should  the  cultivation  of  more  than  one  generation  be  desired,  the  leukocyte 
upper  layer  must  be  carefully  pipetted  off,  as  the  leukocytes  immediately  destroy 
the  merozoites.  Only  the  parasites  within  red  cells  escape  phagocytosis.  Sexual 
parasites  are  much  more  resistant,  and  the  authors  think  they  observed  partheno- 
genesis. The  temperature  should  be  from  40  to  41°  C.  and  strict  anaerobic  condi- 
tions observed,  ^stivo-autumnal  organisms  are  more  resistant  than  benign  tertian 
ones.  Dextrose  seems  to  be  an  essential  for  the  development  of  the  parasites. 

Bass  considers  that  P.  vivax  has  a  flat  amoeboid  like  structure  which  enables  it 
to  squeeze  through  the  brain  capillaries  while  adult  schizonts  of  P.  falciparum  have 
a  solid  oval  form  which  causes  them  to  be  caught  in  the  capillaries. 

Belonging  like  the  malarial  parasite  to  the  Haemosporidia  we  have  a  group  of 
parasites  known  as  the  PIROPLASMS.  The  correct  name  for  these  parasites  is  Babesia 
but  they  are  better  known  under  the  name  Piroplasma.  They  are  minute  organisms, 
usually  pear  or  rod  shape,  which  invade  the  red  corpuscles.  They  produce  no 
pigment  but  destroy  the  corpuscle  and  set  free  the  Hb.  which  is  excreted  in  enor- 
mous amounts  by  the  kidneys.  It  is  this  which  gives  the  name  redwater  to  the  better 
known  Texas  fever  of  cattle.  Organisms  of  this  kind  have  been  thought  of  in  con- 
nection with  blackwater  fever  of  man.  Seidelin  has  claimed  that  a  parasite  of 
similar  nature,  P  ar  a  plasma  flavigemim,  was  the  cause  of  yellow  fever. 

At  one  time  spotted  fever  of  the  Rocky  Mountains  was  supposed  to  be  due  to 
a  parasite  named  Babesia  hominis. 


CHLAMYDOZOA 


243 


SARCOSPORIDIA. 

Sarcosporidia  are  sporozoa  found  in  the  striped  muscles  of  various 
mammals  and  birds.  They  are  common  in  the  pig  and  mouse  and 
have  been  reported  for  man  in  three  well-authenticated  cases.  In  the 
last,  Darling  found  these  protozoa  in  the  biceps  muscle  of  a  negro  patient 
in  Panama.  In  Baraban's  case  the  laryngeal  muscles  at  autopsy  were 
found  to  show  cysts  about  1/15  inch  long  which  contained  sickle-shape 
sporozoites  about  g/*  long. 

They  are  known  also  as  Miescher's  tubes  when  in  mus- 
cle fibers.  They  are  divided  into  three  genera:  Miescheria  and 
Sarcocystis  when  parasitic  in  muscle  fiber;  Balbiania,  when 
parasitic  in  the  in  tervening  connective  tissue  of  the  muscles. 
The  method  of  transmission  is  unknown.  In  some  places  more 
than  50%  of  the  sheep  and  pigs  may  show  infection. 

Miescheria  has  a  thin  membrane  surrounding  the  cyst  while 
that  of  Sarcocystis  is  thickened  and  radially  striated  by  small 
canaliculi. 

As  the  young  trophozoite  grows  nuclei  increase  and  a  definite 
membrane  forms  which  the  sporoblasts  eventually  fill.  Ac- 
cording to  Minchin  the  Sarcosporidia  contain  only  one  genus, 
Sarcocystis.  It  is  never  parasitic  for  invertebrate  hosts  and 
while  occasionally  found  in  birds  and  reptiles  it  is  pre-emin- 
ently a  parasite  of  the  higher  vertebrates.  As  a  rule,  they  are 
harmless  parasites  but  the  Sarcocystis  muris  is  very  pathogenic 
for  the  mouse.  Closely  related  to  the  order  Sarcosporidia  is  the 
parasite  Rhinos poridium  kinealyi. 

Rhinosporidium  kinealyi. — It  causes  pedunculated  tumors  of 
nasal  cavity.  The  pansporoblasts  enlarge  in  the  center  of  the 
connective  tissue  of  the  nasal  polyp  and  contain  about  12 
sporoblasts.  When  mature  the  cystic-like  polyp  bursts  and  the 
sporoblasts  are  liberated  to  extend  the  infection. 


FIG.  63. — Mie- 
scher's sac  from 
the  musculature 
of  a  hog.  X30 
diameters.  (After 
Ostertag.) 


CHLAMYDOZOA. 

These  organisms  are  generally  considered  as  being  protozoal  in 
nature  and  as  a  rule  belong  to  the  filterable  viruses,  which  is  the  desig- 
nation for  the  infectious  principles  of  those  diseases,  in  which  filtration 
of  defibrinated  blood  or  serum  through  a  Berkefeld  filter  capable  of 
holding  back  so  small  an  organism  as  the  M.  melitensis,  does  not  pre- 
vent the  infection  being  transmitted  when  introduced  by  the  proper 
atrium  of  infection.  The  Chlamydozoa  are  also  characterized  by  the 
occurrence  of  "cell  inclusions." 


244  THE   PROTOZOA 

The  best  known  infections  of  this  group  of  diseases  in  man  are  smallpox,  vaccinia, 
rabies,  trachoma,  molluscum  contagiosum,  and  foot  and  mouth  disease.  There  are 
many  such  infections  in  other  animals.  The  cell  inclusions  are  regarded  as  prod- 
ucts of  cellular  reaction  to  a  virus  which  is  more  or  less  impossible  of  demonstra- 
tion. The  discovery  of  exceedingly  minute  granules  in  some  of  these  diseases,  as 
in  variola  and  trachoma,  has  suggested  that,  as  a  reaction  to  the  invasion  by  such 
a  granule,  the  cell  throws  an  enveloping  mantle  about  the  invading  particle.  To 
designate  this  we  use  the  name  Chlamydozoa. 

The  generic  name  Cytorrhyctes  has  been  applied  to  certain  of  these  viruses, 
thus  C.  vaccinias  develops  within  the  epithelial  cells  of  stratified  epithelium.  In 
vaccinia,  Councilman  and  his  colleagues  consider  that  the  development  only  takes 
place  in  the  cytoplasm  of  the  cell.  In  variola,  however,  the  developmental  cycle 
affects  the  nucleus. 

Cytorrhyctes  luis,  reported  as  the  cause  of  syphilis,  sporulates  in  the  blood- 
vessels and  in  the  connective  tissue,  not  in  epithelial  cells. 

Cytorrhyctes  scarlatinas  was  reported  by  Mallory  to  have  been  found  in  the  skin 
in  four  cases  of  scarlet  fever. 


CHAPTER  XVII. 


FLAT  WORMS. 

CLASSIFICATION  OF  THE  PLATYHELMINTHES  (FLAT  WORMS). 


Class 


Family 


Trematoda 


Fasciolidae 


Paramphistomidse 


Schistosomidse 


Genus 

Fasciola 

Fascioletta 

Fasciolopsis 

Dicrocoelium 

Paragonimus 

Opisthorchis 

Clonorchis 

Heterophyes 

Cladorchis 

Gastrodiscus 

Schistosomum 


Cestoda 


,.,        f  Dibothriocephalus 
Dibothnocephahdae     I 


Tseniidae 


Diplogonoporus 
Dipylidium 

Hymenolepis 


Taenia 
Davainea 


Species 

F.  hepatica 
F.  ilocana 

F.  buski 

D.  lanceatum 
P.  westermanii 
O.  felineus 
C.  sinensis 
C.  endemicus 
H.  heterophyes 

C.  watsoni 

G.  hominis 

S.  haematobium 
S.  japonicum 
S.  mansoni 

D.  latus 
D.  grandis 
D.  caninum 
H.  nana 

H.  diminuta 

T.  solium 

T.  saginata 

D.  madagascariensis 


NOTE. — Two  larval  Taeniidaeare  found  in  man  (Cysticercus  cellulosae  and  Echino- 
coccus  polymorphus). 

Also  two  larval  Dibothriocephalidae  (Sparganum  mansoni  and  Sparganum 
prolifer). 

Two  parasites  often  referred  to  as  ophthalmic  flukes  have  been  reported  lying 
between  the  crystalline  lens  and  its  membrane.  They  have  been  considered  as 
possibly  trematode  larvae.  Distomum  ophthalmobium  was  found  in  1850  in  the  eye 
of  a  child  and  Monostoma  lentis  in  the  eye  of  an  old  woman. 

TREMATODES  OR  FLUKES. 

Flukes  are  generally  leaf-like  in  outline,  rarely  cylindrical,  and  exhibit 
marked  variation  in  size  and  shape.  They  are  nonsegmented  and  do 

245 


246  FLAT   WORMS 

not  have  cilia  on  ectoderm.  Very  characteristic  of  them  is  the  posses- 
sion of  suckers  by  which  they  hold  on  to  the  skin  or  alimentary  system  of 
their  host. 

They  are  divided  into  two  orders:  i.  the  Monogenea  in  which  the  egg  gives  rise 
to  a  larva  which  later  becomes  the  adult  and  2.  the  Digenea.  It  is  to  this  latter  that 
the  flukes  parasitic  in  man  belong.  This  order  is  characterized  by  the  fact  that  the 
larva  becomes  parasitic  in  some  second  animal  and  then  gives  rise  to  a  second  gen- 
eration of  larvae  which  latter  develop  into  adults. 

The  largest  human  fluke,  Fasciolopsis  buski,  is  from  two  to  three  inches  (50  to 
75  mm.)  in  length,  while  the  Heterophyes  heterophyes  is  less  than  1/12  of  an  inch 
(2  mm.)  in  length.  The  most  important  fluke,  the  liver  fluke,  Clonorchis  endemicus, 
is  flat  and  almost  transparent,  while  the  almost  equally  important  lung  fluke,  the 
Paragonimus  westermanii,  is  oval,  almost  round  and  reddish-brown  in  color.  With 
the  exception  of  the  Schistosomidse,  all  flukes  are  hermaphrodites,  and,  with  the 
exception  of  this  family,  all  flukes  have  operculated  eggs.  The  only  other  opercu- 
lated  (with  a  lid)  eggs  we  meet  with  in  man  are  those  of  the  Dibothriocephalidae. 

The  three  important  families  of  flukes  parasitic  for  man  are:  i. 
Paramphistomidas — flukes  with  two  suckers  situated  at  either  extremity. 
2.  Fasciolidae — -flukes  with  two  suckers,  one  terminal,  the  other  adjacent 
to  it  and  situated  ventrally.  This  family  includes  the  important  geneia 
Fasciola,  Opisthorchis,  Dicroccelium,  Fasciolopsis,  and  Paragonimus. 
In  Paragonimus  and  Heterophyes  the  genital  pore  is  posterior  to  the 
acetabulum,  in  the  other  genera  it  is  anterior.  Fasciola  has  a  den- 
dritic intestinal  canal  which  is  not  the  case  with  Clonorchis,  Fascio- 
lopsis, Fascioletta,  Opisthorchis  and  Dicroccelium.  In  Dicroccelium 
the  testicles  are  anterior  to  the  uterus,  in  Opisthorchis,  Clonorchis, 
Fasciolopsis  and  Fascioletta  they  are  posterior.  Fasciolopsis  and 
Clonorchis  have  branched  testicles  (the  former  a  very  large  fluke- 
Clonorchis  of  medium  size)  while  those  of  Opisthorchis  are  lobed. 

3.  Schistosomidae :  In  this  family  we  have  a  leaf-like  male  which  by 
a  folding  in  of  its  sides  makes  a  channel  for  the  thread-like  female.  The 
sexes  are  separate,  not  hermaphroditic  as  with  the  Fasciolidae  and 
Paramaphistomidae. 

Flukes  have  two  suckers  which,  except  in  the  Paramphistomidae,  are  quite  near 
each  other — one  is  termed  the  oral  sucker  and  the  other  the  ventral  sucker  or  acetabu- 
lum. The  intestinal  tract  consists  of  a  pharynx,  proceeding  from  the  oral  sucker, 
which  bifurcates  and  terminates  in  blind  intestinal  casca. 

At  the  posterior  extremity  is  an  excretory  pore  which  is  at  the  termination  of 
a  duct  which  divides  into  ramifying  branches.  This  is  the  water-vascular  system. 
The  testes,  of  various  shapes  and  relations  to  the  uterus,  are  more  or  less  centrally 
situated  and  have  vasa  deferentia.  In  some  flukes  the  receptaculum  seminis  is  a 


FLUKES  247 

conspicuous  organ.  The  vitellaria  are  bilateral  branching  glands  which  pour 
nutrient  material  into  the  ootype.  It  is  in  the  ootype  that  the  eggs  are  formed, 
and  opening  into  it  we  have  the  adjacent  ovary.  The  shell  gland  is  near  the  ovary. 

A  canal,  known  as  Laurer's  canal,  leads  from  the  ootype  to  the  exterior,  the 
function  of  which  is  in  question.  It  is  probable  that  as  trematodes  have  no  sperma- 
theca,  the  spermatozoa  from  other  flukes  enter  by  way  of  this  canal.  The  life  his- 
tory of  the  important  human  flukes  is  unknown.  It  is  supposed  that  this,  in  a  meas- 
ure, may  resemble  that  of  the  common  liver-fluke  of  sheep  (sheep  rot).  In  this  the 
eggs  containing  a  ciliated  embryo  (miracidium)  pass  out  in  the  faeces.  This  embryo 
is  hatched  out  and,  gaining  the  water,  swims  about  actively  until  it  reaches  some 
suitable  mollusk  (Limnaea  truncatula).  By  means  of  a  pointed  end,  it  bores  its 
way  into  the  body  of  the  gasteropod  and  in  the  pulmonary  chamber  becomes  a 
bag-like  structure  (the  sporocyst)  from  the  germinal  cells  of  which  develop  a  creature 
with  an  alimentary  canal  (redia).  The  rediae  tend  to  break  out  of  the  sporocyst  and 
wander  to  the  liver  of  the  snail.  These  rediae  may  give  rise  to  a  second  generation 
of  rediae. 

From  the  rediae  minute  little  worms  resembling  adult  flukes  in  possessing  suckers, 
but  differing  in  the  possession  of  a  tail,  develop  (cercaria).  Having  reached  maturity, 
these  cercariae  leave  the  rediae,  and,  as  in  case  of  Fasciola  hepatica,  lose  the  tail,  be- 
come encysted  on  blades  of  grass,  to  be  eaten  by  sheep  and  again  commence  the  cycle. 
The  encysted  cercariae  develop  into  adult  liver  flukes.  It  is  probable  that  with  many 
flukes  the  cercarise  enter  some  host,  as  mollusk,  insect,  or  fish,  and  that  it  is  by  eat- 
ing such  animals  as  food  that  man  becomes  infected.  Looss  thinks  it  possible  that 
the  miracidium  of  Schistosomum  haematobium  may  bore  its  way  directly  into  man, 
as  do  the  larvae  of  the  hookworm.  Manson  also  suggests  that  the  reporting  by  Mus- 
grave  of  100  mature  lung  flukes  in  a  psoas  abscess  makes  it  very  probable  that  these 
parasites  entered  the  body  as  miracidia.  The  idea  in  China  is  that  the  infection 
with  the  common  liver  fluke  of  man  is  brought  about  by  eating  fish.  Fluke  disease 
is  generally  known  as  distomatosis  or  distomiasis. 


LIVER  FLUKES. 

Fasciola  hepatica  (Distomum  hepaticum). — This  fluke,  while  of 
enormous  economic  importance  by  reason  of  destruction  of  sheep,  has 
only  been  reported  twenty-three  times  in  man,  and  in  these  instances 
does  not  seem  to  have  occasioned  marked  symptoms. 

It  has  a  cone-shaped  anterior  projection  and  is  about  11/4  inch  (30  mm.)  long. 
The  intestinal  canal,  as  well  as  the  testicles,  is  branched.  There  is,  however,  a  possible 
importance  of  F.  hepatica  in  connection  with  a  peculiar  affection  known  as  "halzoun. " 
This  results  from  the  eating  of  raw  goat-liver,  and  it  is  supposed  that  the  flukes 
crawl  up  from  the  stomach  and,  entering  the  larynx  or  attaching  themselves  about 
the  glottis,  produce  the  asphyxia  characteristic  of  the  disease. 

Dicrocoslium  lanceatum. — This  has  only  been  reported  seven  times  in  man. 
The  symptoms  are  unimportant.  The  fluke  is  about  1/3  of  an  inch  (8  mm.)  long, 
with  testicles  anterior  to  the  uterus. 


248 


FLAT   WORMS 


Clonorchis  endemicus  (Opisthorchis  sinensis).— This  fluke  and  the 
C.  sinensis  are  the  most  important  of  the  human  liver  flukes.  Until 
recently  these  flukes  were  known  as  Opisthorchis  sinensis. 

Looss  has  separated  this  genus  from  Opisthorchis  principally  by  the  character- 
istic of  branching  testicles — those  of  Opisthorchis  being  lobed.  This  fluke  is  very 
common  in  China  and  Japan — in  certain  sections  of  Japan  20%  of  the  population 
being  infected.  This  fluke  is  about  1/4  to  1/2  inch  (8  mm.)  long  and  C.  sinensis 
about  3/4  of  an  inch  long  and  1/6  of  an  inch  broad  (16  X  4  mm.)  When  squeezed 


FIG.  64. — Trematodes  of  man,  natural  size,  i,  Clonorchis  endemicus  (Opisthor- 
chis sinensis);  2,  Gastrodiscus  hominis;  3,  Dicrocoelium  lanceatum;  4,  Heterophyes 
heterophyes;  5,  Schistosomum  haematobium;  6,  Fasciola  hepatica;  7,  Paragonimus 
westermanii;  8,  Fasciolopsis  buski;  9,  Opisthorchis  felineus;  10,  anatomy  of  C.  en- 
demicus (enlarged).  G.  P.,  genital  pore;  V.  S.,  ventral  sucker;  V.  G.,  vitelline  glands; 
R.  S.,  receptaculum  seminis;  T.,  branched  testicles. 

out  of  the  thickened  bile  ducts  it  is  so  transparent  and  glairy  as  almost  to  resemble 
glairy  mucus.  As  many  as  4000  of  these  parasites  have  been  found  in  a  case,  chiefly 
in  the  liver,  but  at  times  in  the  pancreas.  This  fluke  is  supposed  to  produce  most 
serious  symptoms,  as  indigestion,  swelling  and  tenderness  of  liver,  ascites,  oedema, 
and  a  fatal  cachexia.  As  a  matter  of  fact,  many  physicians  in  China  attribute  very 
little  pathogenic  importance  to  it.  The  disease  is  diagnosed  by  the  presence  of  the 
ova  in  the  stools.  The  source  of  infection  is  probably  through  the  eating  of  uncooked 
fish. 

Kobayashi  has  examined  various  mollusks  and  fish  for  trematode  larvae.     He 


FLUKES  249 

succeeded  in  infecting  nine  kittens  and  two  cats  by  feeding  them  with  certain  fresh- 
water fishes  whose  flesh  contained  trematode  larvae.  These  fish  were  found  in 
districts  where  human  distomiasis  was  common.  The  view  is  taken  that  the  two 
species  of  Clonorchis  are  identical. 

Opisthorchis  felineus. — This  fluke  is  smaller  than  the  C.  endemicus,  and  is  a 
common  parasite  of  the  gall  bladder  and  bile  ducts  of  cats.  There  are  two  lobed 
testicles  in  this  species  instead  of  dendritic  ones  as  in  C.  endemicus.  In  certain 
parts  of  Siberia  the  parasite  is  found  in  more  than  6%  of  the  human  autopsies.  The 
symptoms  are  similar  to  those  caused  by  C.  endemicus. 

Other  liver  flukes  of  less  importance  which  have  been  reported  for  man  are: 
i.  Opisthorchis  noverca.  This  was  found  in  bile  ducts  of  two  natives  of  Calcutta. 
It  was  lancet-shaped  and  covered  with  spines. 

2.  Metorchis  truncatus:  This  is  a  small  fluke,  1/12  inch  (2  mm.)  long,  squarely 
cut  across  at  its  posterior  end  and  covered  with  spines.  This  was  possibly  found 
once  in  man. 

Intestinal  Flukes. 

Cladorchis  watsoni  (Amphistomum  watsoni). — This  fluke  is  about  1/3  of  an 
inch  (8  mm. )  long,  of  oval  outline  but  broader  at  posterior  end  and  has  an  indistinct 
oral  sucker  and  a  large  sucker  at  the  other  end.  This  parasite  has  only  been  reported 
once.  Eggs,  125  X  75/*- 

Gastrodiscus  hominis  (Amphistomum  hominis). — This  fluke  is  about  1/4  of  an 
inch  (6  mm.)  long  and  has  a  disc-like  acetabulum  about  1/6  of  an  inch  in  diameter 
from  which  proceeds  a  teat-like  projection,  bearing  an  oral  sucker.  While  it  has 
only  been  reported  twice  for  man,  indications  are  that  it  is  probably  fairly  common 
in  India  and  Assam.  Eggs,  150  X  72fi. 

Fasciolopsis  buski  (Distomum  crassum). — This  is  probably  a  rather  common 
parasite  in  India,  as  Dobson  found  the  eggs  in  i%  of  the  stools  of  more  than  1000 
coolies.  The  fluke  is  from  2  to  3  inches  (40  to  70  mm.)  in  length  and  about  1/2  of  an 
inch  (12  mm.)  in  breadth.  It  is  thick,  brown  in  color,  and  has  a  very  large  acetabu- 
lum, three  times  the  size  of  the  oral  sucker  and  located  almost  adjacent  to  it.  The 
branched  ovary  and  shell  gland  lie  in  the  center  with  the  branched  testicles  posterior. 
The  coiled  uterus  is  anterior  to  the  testicles.  Eggs,  125  X  75j".  These  parasites 
cause  dyspeptic  symptoms  and  an  irregular  diarrhoea.  It  is  also  called  Distomum 
crassum.  F.  rathouisi  is  now  considered  to  have  been  a  shrunken  F.  buski,  as  it 
seems  to  be  anatomically  similar  to  F.  buski.  Kwan's  fluke  reported  from  Hong 
Kong,  was  possibly  F.  buski. 

Heterophyes  heterophyes  (Cotylogonimus  heterophyes). — This  exceedingly 
small  fluke  (2  X  0.5  mm.),  which  can  be  recognized  by  its  small  size  (less  than  1/12 
of  an  inch  long)  and  large,  prominent  acetabulum,  was  formerly  supposed  to  be  rare. 
The  oral  sucker  is  much  smaller  than  the  acetabulum.  The  elliptical  testicles  lie 
at  the  extreme  posterior  end.  Cuticle  has  scale-like  spines.  The  eggs  are  30X1 7/*. 
Very  characteristic  of  this  genus  is  the  large  sucker-like  genital  pore  just  below  and 
to  one  side  of  the  acetah^ulum.  Looss  has  shown  that  it  is  quite  common  in  Egypt, 
he  having  found  it  twice  in  Alexandria  in  nine  autopsies.  The  parasites  occupy  the 
ileum.  It  is  common  in  dogs. 


250 


FLAT   WORMS 


Fascioletta  ilocana. — This  is  a  small  fluke,  about  1/4  inch  (6  mm.)  long.  There 
are  two  massive  testicles  in  the  posterior  part  of  body.  The  acetabulum  is  promi. 
nent.  The  egg  of  this  small  fluke  is  quite  large  (ioc/0  and  has  an  operculum. 
These  trematodes  were  found  by  Garrison  in  five  natives  of  Luzon,  P.  I.,  after  treat- 
ment with  male  fern. 


FIG.  65. — Anatomy  of  a  tape-worm,  Tasnia  solium  (A.,  longitudinal,  B.,  cross 
section);  a  fluke,  Paragonimus  westermanii  (C).,  male  and  female  nematode,  Oxyuris 
vermicularis  (D.).  A.  i,  Testes;  2,  yolk  glands;  3,  shell  glands;  4,  ovary;  5,  vagina; 

6,  vas  deferens;  7,  uterus  before  branching;  8,  water-vascular  system.     B.  i,  Cuticle; 
2,  circular  muscle;  3,  ovary;  4,  testes;  5,  uterus;  6,  excretory  canal;  7,  nerve  cord. 
C.   i,  Oral  sucker;  2,  acetabulum;  3,  uterus;  4,  testes;  5,  excretory  canal;  6,  ovary; 

7,  yolk  glands.     I),  (a)  Female,     i,  Vulva;  2,  uterus;  3,  bulb  of  oesophagus;  4,  anus; 
(b)  Male,     i,  Bulbous  mouth  end;  2,  testes;  3,  spicule;  4,  alimentary  canal.     E. 
Egg  of  P.  westermanii. 


LUNG  FLUKES. 

Paragonimus  westermanii  (Distoma  ringeri).— In  certain  parts  of 
Japan  and  Formosa  it  is  estimated  that  as  many  as  10%  of  the  inhab- 
itants may  harbor  this  parasite. 

It  is  also  common  in  China,  and  recently  many  cases  have  been  reported  in  the 
Philippines.  Dr.  Stiles  states  that  around  Cincinnati,  Ohio,  there  was  at  one  time 
quite  a  heavy  infection  among  the  hogs,  so  that  it  may  be  that  certain  cases  diagnosed 
in  man  as  pulmonary  tuberculosis  are  paragonimiasis. 


BILHARZIASIS  251 

It  is  popularly  known  as  endemic  haemoptysis  on  account  of  the  accompanying 
symptoms  of  chronic  cough  and  expectoration  of  a  rusty-brown  sputum.  After 
violent  exertion,  and  at  times  without  manifest  reason,  attacks  of  haemoptysis  of 
varying  degrees  of  severity  come  on.  The  characteristic  ova  are  constant  in  the 
sputum  and  establish  the  diagnosis.  The  fluke  itself  is  a  little  more  than  1/3  of  an 
inch  (8  mm.)  long  and  is  almost  round  on  transverse  section,  there  being,  however, 
some  flattening  of  the  ventral  surface.  The  acetabulum  is  conspicuous  and  opens 
just  anterior  to  the  middle  of  the  ventral  surface.  Eggs  about  90  x  65  /*. 

The  branched  testicles  are  posterior  to  the  laterally  placed  uterus  and  the 
genital  pore  opens  below  the  acetabulum.  The  branched  ovary  is  opposite  the  uterus 
on  the  other  side. 

It  is  rather  flesh-like  in  appearance  and  is  covered  with  scale-like  spines.  The 
flukes  are  usually  found  in  tunnels  in  the  lungs,  the  walls  of  which  are  of  thickened 
connective  tissue.  There  may  be  also  cysts  formed  from  the  breaking  down  of 
adjacent  tunnel  walls.  In  addition  to  lung  infection  with  this  fluke,  brain,  liver, 
and  intestinal  infections  may  be  found.  Musgrave  was  the  first  one  to  call  attention 
to  the  frequency  of  general  infection  with  this  parasite  (paragonimiasis)  in  the 
Philippines.  He  found  it  in  seventeen  cases  in  one  year.  The  life  history,  beyond 
the  stage  of  miracidium,  is  unknown. 

Another  fluke  which  has  been  reported  from  the  lung  is  Fasciola  gigantea  (very 
similar  to  F.  hepatica).  This  was  coughed  up  by  a  French  officer  who  had  been  in 
Africa. 


BLOOD  FLUKES. 

Schistosomum  haematobiuin. — Flukes  of  the  circulatory  system 
are  of  great  importance  in  Egypt,  South  Africa,  Japan,  and  the  West 
Indies.  The  disease  is  named  bilharziasis  after  Bilharz  who  in  1851 
first  associated  the  parasite  and  the  disease. 

It  seems  probable  that  there  are  at  least  three  human  species,  differentiated 
principally  by  the  appearance  of  the  egg.  In  the  blood-fluke  disease  of  Egypt, 
(S.  haematobium),  the  parasite  chiefly  infects  the  bladder  and  the  egg  has  a  terminal 
spine.  The  terminal-spined  ovum  is  also  found  in  the  rectum  and  in  the  faeces.  In 
the  West  Indies,  as  shown  by  the  reports  of  Surgeon  Holcomb  from  Porto  Rico, 
rectal  bilharziasis  is  rather  common.  In  these  cases  the  egg  is  practically  always 
lateral-spined.  Looss  thinks  that  the  lateral-spined  egg  is  the  product  of  an  unfer- 
tilized female  S.  haematobium.  These  flukes  differ  from  other  human  flukes  in 
possessing  nonoperculated  eggs  as  well  as  in  having  the  sexes  separate.  The  adults 
of  this  species,  the  S.  mansoni,  are  scarcely,  if  at  all,  to  be  distinguished  from  the 
S.  haematobium.  Leiper  has  recently  noted  a  difference  in  that  the  male  of  S. 
mansoni  has  7  testicles  as  against  4  for  S.  haematobium.  With  S.  japonicum,  the 
name  of  the  Eastern  species,  there  is  not  only  the  difference  that  the  eggs  are  without 
spines,  but,  in  addition,  the  skin  of  the  adult  parasite  is  not  tuberculated,  as  is  the 
case  with  the  other  two  species.  It  is  slightly  smaller,  the  acetabulum  projects  more 
prominently,  and  the  lower  part  of  the  male  infolds  more  markedly  than  in  S. 


FLAT   WORMS 

haematobium.  Catto  considers  that  the  S.  japonicum  may  live  in  both  arteries  and 
veins.  The  other  two  species  only  live  in  branches  of  the  portal  vein.  The  blood 
flukes  are  about  1/2  inch  (13  mm.)  long.  All  of  these  flukes  live  separately  until 
maturity.  At  this  time  the  female  enters  what  is  known  as  the  gynaecophoric  canal 
of  the  male;  this  canal  is  formed  by  the  infolding  of  the  sides  of  the  flat  male  fluke, 
thus  giving  a  rounded  appearance  to  the  male.  The  female  is  longer  than  the  male 
(about  5/6  of  an  inch  long),  and  is  thread-like  and  of  a  darker  color.  Her  two 
extremities  project  from  the  canal  of  the  male  in  which  she  lives. 

The  oral  sucker  of  the  male  is  infundibuliform  and  is  smaller  than  the  peduncu- 
lated  acetabulum.  In  the  female  the  oral  sucker  is  larger  than  the  acetabulum. 
The  eggs  are  fusiform,  yellowish  in  color,  have  a  thin  shell  and  a  terminal  spine. 

The  most  prominent  symptoms  of  the  Bilharz  disease  are  haemat- 
uria  and  bladder  irritation;  later  on  calculus  formation.  In  rectal 
bilharziasis  the  symptoms  are  more  those  of  bleeding  piles  or  of  a  mild 
dysentery. 

There  may  also  be  involvement  of  the  appendix.  In  the  Japanese  infection 
the  symptoms  point  more  to  liver  and  spleen,  there  being  ascites,  cachexia,  and  a 
bloody  diarrhoea. 

The  eggs  of  the  S.  japonicum  are  readily  found  in  the  fasces;  they  are  about 
100  X  70/4.  They  are  oval,  transparent,  and  with  a  smooth  shell,  within  which  can 
be  made  out  the  outlines  of  an  embryo.  Upon  adding  water  the  ciliated  embryo 
begins  to  show  movement  in  about  ten  minutes  and  shortly  afterward  bursts  out 
of  the  shell  and  swims  about  actively.  It  is  more  melon-shaped  than  the  miracidium 
of  S.  haematobium. 

The  life  history  is  not  known  of  any  of  these  flukes.  Looss  conjectures  that  it 
is  probable  that  the  miracidium  enters  the  skin,  not  requiring  an  intermediary  host. 
Frequent  experiments  have  failed  to  show  any  mollusk,  etc.,  which  attracted  the 
embryo.  Evidence  seems  to  show  that  those  who  are  constantly  wading  about  in  the 
water  of  the  pools  or  the  mud  of  the  fields  are  the  ones  most  subjected  to  infection. 

Katsurada,  by  experiments  with  a  cat  and  dog,  has  proved  that  infection  will 
take  place  through  the  shaved  skin  of  an  animal  held  in  infected  water — none  of 
the  water  being  allowed  to  enter  by  mouth.  Fully  developed  miracidia  and  male 
and  female  flukes  were  found  in  the  portal  vein.  It  is  thought  that  further  develop- 
ment of  the  miracidia  in  the  body  may  account  for  the  heavy  infection. 

Turner  has  recently  noted  the  frequency  of  bilharzial  affections  of  the  lungs  in 
South  Africa  (50%  in  natives)  and  he  thinks  this  may  be  an  important  factor  in 
prevalence  of  lung  diseases  in  the  natives  of  this  region.  He  considers  bathing  in 
contaminated  waters  of  prime  importance  in  the  causation  of  the  infection  which 
he  thinks  is  probably  by  way  of  the  skin. 

A  recent  view  is  that  the  miracidium  enters  while  bathing  by  the  preputial 
channel,  hence  the  value  of  circumcision. 

If  urine  containing  eggs  is  diluted  with  water  the  miracidium  breaks  out  of  the 
shell  and  swims  about  as  if  in  search  of  some  desired  object. 

The  view  is  also  entertained  that  the  miracidium  may  gain  access  to  the  body 
through  the  drinking  water;  there  is  much  evidence  against  this.  However  access 


TAPE   WORMS 


253 


to  the  body  is  gained,  it  is  known  that  the  larval  forms  make  their  way  to  the  liver 
where  they  develop.  Arriving  at  maturity,  the  males  and  females  become  united 
and  proceed  to  the  terminal  branches  of  the  portal  vein,  where  the  irritating  eggs, 
given  off  by  the  female,  give  rise  to  the  symptoms. 


Ova  or  the  Parasitic  Worms  or  Man  • 
TREMATODA 


N  TO  SCALE  X      ICOO 


Heterophyes 
heterophyes 

(after  Loo*S.IQO,'5)  ,«y 


Di 
coe 

l&ncetxtum 


Opisthorchis 
felineus^ne 

Clqnorcliis  Clonorchis 
sihensis  endemicus 

(Nodi:i.-il  fiui:.  LOOM.IW) 


Fasciola  Fasciolopsis 

heptxtica      buskii  (*^^ 

{n.f\crtoo«s.l905) 


FIG.  66. — Trematode  ova. 


CESTODE  OR  TAPE-WORM  INFECTIONS. 

The  cestodes  and  trematodes  constitute  the  two  great  divisions  of 
the  flat  worms.  Anatomically,  a  tape-worm  may  be  considered  as  a 
series  of  individual  flukes  united  in  one  ribbon-like  colony.  The 
cestode  segments,  or  proglottides  are  covered  by  an  elastic  cuticle  and 
in  their  interior  usually  contain  striated  elliptical  bodies  composed  of 
calcium  carbonate  about  5  to  25/1  according  to  the  species  in  which  they 
are  found. 

These  calcareous  bodies  are  characteristic  of  cestode  tissue.  They  have  been 
mistaken  for  coccidia.  There  is  no  mouth  or  alimentary  canal  in  tape-worms,  the 
segments  absorbing  their  nourishment  through  the  general  surface. 


254 


FLAT   WORMS 


A  tape-worm  is  divided  into  the  segment-producing  controlling  head  and  the 
series  of  segments  or  proglottides  together  known  as  the  strobila.  The  head  and  neck 
together  form  the  scolex.  Tape-worm  heads  are  provided  with  suctorial  or  hook- 
like  organs,  or  both,  to  enable  them  to  hold  on  to  the  intestinal  mucosa. 

The  hooks  when  present  on  the  anterior  extremity  of  the  head  are  carried  by  a 
protrusible  structure  called  the  rostellum. 

The  importance  of  the  head  is  generally  recognized  by  the  well- 
known  fact  that  the  permanent  evacuation  of  one  of  these  parasites  is 
only  arrived  at  when  the  head  as  well  as  the  segments  is  expelled. 
Otherwise,  additional  segments  will  be  produced. 


10 


7-0% 


FIG.  67. — Tape-worms.  A.  i,  2  and  3,  Scolex,  proglottides  and  ovum  of  Taenia 
solium;  B.  4,  5,  6  and  7,  Scolex,  prologlottides  and  ovum  of  Dibothriocephalus  latus; 
C.  8,  9,  and  10,  Scolex,  proglottides  and  ovum  of  Taenia  saginata.  The  onchosphere 
in  10  is  shown  within  the  outer  yelk  coating  (frequently  seen  in  stools).  In  3  only 
the  onchosphere  within  the  embryonal  shell  is  shown. 

Even  in  tape-worms  twenty-five  to  thirty  feet  in  length,  the  head  is  no  larger 
than  a  small  shot.  It  carries  the  suckers  or  hooklets  which  best  enable  us  to  differ- 
entiate the  different  species.  The  segments  adjacent  to  the  head  are  immature — 
the  sexually-mature  ones  being  found  from  the  middle  of  the  body  onward.  The 
sexually-mature  segment  possesses  a  varying  number  of  testicles:  three  in  Hymeno- 
lepis  nana  and  as  many  as  2000  in  Taenia  saginata.  As  with  the  flukes,  they  also 
have  vasa  deferentia,  cirrus,  ovaries,  yolk  glands,  uterus,  genital  pore,  etc.  The 


TAPE   WORMS  255 

location  of  the  genital  pore  and  the  character  of  the  branching  of  the  uterus  are  of 
the  greatest  importance  in  differentiation.  The  sexually-mature  proglottides  may 
either  expel  their  ova,  when  these  would  be  found  in  the  faeces  or,  as  is  common, 
they  break  off  and  pass  out  themselves  in  the  faeces.  Then  they  either  expel  the 
eggs  or  may  be  eaten  by  some  animal  and  in  this  way  effect  an  entrance  for  their  ova. 
It  is  an  important  practical  point  that  the  fasces  of  a  patient  with  T.  solium  or  T. 
saginatamay  not  show  any  ova,  these  passing  out  in  the  intact  segments.  The  oval 
operculated  eggs  of  Dibothriocephalus  latus,  however,  are  constantly  in  the  faeces. 
The  "hexacanth"  or  six-hooked  embryo,-also  called  the  onchosphere,  is  the  essen- 
tial part  of  the  egg.  The  embryonic  envelope  is  dissolved  off  in  the  alimentary 
canal  of  the  animal  ingesting  it,  and  the  onchosphere  bores  its  way  through  the  gut 
to  later  become  encysted  in  various  tissues.  In  some  tape-worms  a  ciliated  embryo 
is  liberated  from  the  egg  shell  and  swims  about  actively  to  enter  some  fish  or  other 
animal.  When  the  six-hooked  embryo  reaches  its  proper  tissue,  the  hooklets  are 
discarded  and  a  scolex  similar  to  the  parent  one  is  developed.  At  this  time  we  have 
a  bladder-like  structure  with  the  scolex  inverted  in  it.  This  is  termed  the  proscolex 
stage.  This  little  cyst  with  its  scolex  when  ingested  by  another  animal  is  digested, 
and  the  scolex,  establishing  itself  in  the  intestine,  develops  a  series  of  segments. 
The  ciliated  embryo  of  the  D.  latus  does  not  form  a  cyst,  but  instead  a  worm-like 
creature  similar  to  the  adult.  This  is  termed  a  Plerocercoid. 

If  the  larval  stage  shows  a  single  cyst  and  a  single  head,  it  is  termed 
Cysticercus;  if  multiple  cysts  but  only  one  head  to  each  cyst,  Ccenurus; 
while  with  multiple  cysts  and  multiple  heads  in  each  cyst  the  term 
Echinococcus  is  used. 

Where  there  is  very  little  fluid  in  the  cyst  and  the  larva  is  of  minute 
size,  as  with  the  Hymenolepis,  the  term  Cercocystis  is  employed. 


KEY  TO  CESTODE  GENERA. 

I.  Head  with  two  elongated   slit-like   suckers — Genital   pores  ventral — Rosette 
uterus.     Dibothriocephalidcz. 

(A)  Single  set  of  genital  organs  in  each  segment.     Dibothriocephalus. 

(B)  Double  set  of  genital  organs  in  each  segment.     Diplogonoporus. 

(C)  Immature  fofms  showing  characteristics  of  Dibothriocephalidse—  (collective 
group).     Sparganum. 

II.  Head  with  four  cup-like  suckers;  genital  pores  lateral.     T&niida. 

(A )  Uterus  with  median  stem  and  a  varying  number  of  lateral  branches.     Tania. 

(B)  Uterus  without  median  stem  and  lateral  branches. 

(1)  Genital  pores  single.     Rostellum  with  not  more  than  two  rows  of  hooks. 

(a)  Suckers   armed  with   numerous  small  hooklets.     Fifteen   to   twenty 
testicles  in  each  segment.     Davainea. 

(b)  Suckers  not  armed.     Three  testicles  in  each  segment.     Hymenolepsis. 

(2)  Genital  pores   double.     Rostellum   with   four    or   five  rows  of  hooks. 
Dipylidium. 


256  FLAT   WORMS 


INFECTIONS. 

Taenia  saginata  (Tsenia  mediocanellata).—  This  very  widely  dis- 
tributed tape-worm  is  often  termed  the  unarmed  tape-worm,  to  dis- 
tinguish it  from  the  T.  solium  or  armed  tape-worm. 

It  is  from  10  to  25  feet  long  and  has  several  hundred  proglottides.  The  small 
pear-shaped  head  has  four  pigmented  elliptical  suckers  and  no  hooklets.  The  seg- 
ments are  plumper  than  those  of  T.  solium,  hence  the  name  saginata.  The  single 
lateral  genital  pore  projects  markedly  and  in  a  series  of  segments  presents,  as  a  rule, 
first  on  one  side,  and  then  on  the  opposite  side  of  the  next  segment  (alternating). 
The  best  way  to  distinguish  a  segment  of  the  T.  saginata  from  the  T.  solium  is  by 
counting  the  number  of  lateral  uterine  branches;  these  number  fifteen  to  thirty,  are 
quite  delicate  and  branch  dichotomously.  The  lateral  divisions  of  the  uterus  of 
the  T.  solium  are  tree-like  in  their  branching  and  only  number  five  to  twelve  on  each 
side. 

T.  solium  has  three  ovaries  while  T.  saginata  has  only  two.  The  ox  is  the  inter- 
mediate host.  The  eggs  of  Taenia  have  an  oval  outer  shell  which  is  filled  with 
rather  translucent,  refractile  yolk,  often  in  globules.  Within  the  oval  shell  is  the  more 
rounded  cell  of  the  six-hooked  embryo  with  its  thick  striated  membrane.  The  outer 
shell  is  often  absent  in  the  eggs  found  in  the  faeces,  only  the  shell  of  the  six-hooked 
embryo  being  found.  The  six-hooked  embryo,  having  worked  its  way  from  the 
alimentary  canal  to  the  muscles  or  liver  of  the  ox,  becomes  encysted  (Cysticercus 
bovis).  This  little  bladder-like  structure  is  about  1/4  by  1/3  inches,  and  contains 
but  a  small  amount  of  fluid.  Being  ingested  by  man's  eating  raw  or  imperfectly 
cooked  meat,  the  adult  stage  becomes  established  in  his  alimentary  canal. 

It  is  probable  that  the  various  raw-meat  cures  have  made  the  infection  more  com- 
mon. In  Abyssinia  the  infection  is  said  to  be  universal,  and  a  man  without  a  tape- 
worm to  be  a  freak.  An  important  point  is  the  fact  that  the  larval  stage  almost 
never  appears  in  man.  It  is  this  fact  which  makes  it  a  so  much  less  dangerous  para- 
site than  the  T.  solium,  which  readily  establishes  a  larval  existence  in  man  if  the  ova 
are  introduced  into  the  human  stomach.  Cooking  meat  always  destroys  the 
cysticercus.  A  period  of  about  two  months  elapses  after  the  ingestion  of  the  cysti- 
cercus  before  the  mature  segments  pass  out  of  the  rectum.  These  not  only  make 
their  exit  with  the  faeces,  but  are  also  capable  of  wandering  out  at  other  times.  In 
this  they  differ  from  the  segments  of  T.  solium.  T.  saginata  next  to  Hymenolepis 
nana  is  the  common  tape-worm  of  the  United  States.  Dr.  Stiles  has  examined 
several  hundred  tape-worms  in  the  United  States  during  the  past  few  years  and 
has  found  only  one  T.  solium. 

Abnormalities  of  the  scolex  and  proglottides  are  not  uncommon  with  T.  saginata. 
This  is  less  frequently  the  case  with  T.  solium. 

Tsenia  solium.  —  -The  measly-pork  tape-worm  is  smaller  than  the 
T.  saginata  and  differs  from  it  in  having  a  globular  head,  with  a  rostellum 
which  is  crowned  by  twenty-six  to  twenty-eight  hooklets. 

In  T.  saginata  a  depression  takes  the  place  of  the  armed  rostellum;  the  suckers 


DWARF    TAPE    WORM  257 

of  T.  saginata  are,  however,  much  more  powerful  than  those  of  T.  solium.  The 
segments  have  only  five  to  ten  coarse  branches  and  are  expelled  only  at  the  time  of 
defecation.  The  segments  or  the  ova  having  been  ingested  by  a  hog,  the  six-hooked 
embryo  is  liberated  and  becomes  encysted  chiefly  in  the  tongue,  neck,  and  shoulder 
muscles  of  the  hog,  as  an  invaginated  scolex.  Pork  containing  this  cysticercus 
(Cysticercus  cellulosae)  is  known  as  measly  pork.  This  cysticercus  contains  much 
more  fluid  than  that  of  the  ox  and  is  from  1/4  to  4/5  of  an  inch  long.  If  one  by 
chance  should  carry  the  egg  on  his  fingers  to  his  mouth,  as  the  result  of  examining 
mature  segments,  the  larval  stage  may  be  established  in  man.  If  this  infection  is 
not  heavy,  very  few  symptoms  may  be  observed.  The  cysticercus,  however,  tends 
to  invade  the  brain,  next  in  frequency  the  eye,  and  so  causes  convulsions,  death  or 
blindness.  Instead  of  only  being  the  size  of  a  pea,  these  cysts,  when  forming  in  the 
brain,  may  be  the  size  of  a  walnut  or  larger.  T.  solium  is  comparatively  common  in 
North  Germany,  but  is  exceedingly  rare  in  England  and  the  United  States. 

Taenia  africana. — This  is  an  unarmed  tape-worm,  only  about  5  feet  long.  It 
was  found  in  a  native  soldier  in  German  East  Africa.  - 

Garrison  has  reported  from  the  Philippines  a  tape-worm  with  an  unarmed 
rostellum,  V-shape  and  spiral  formation  of  the  uterine  stem  with  compact  structure 
of  the  gravid  uterus  under  the  name  of  Taenia  philippina.  Another  tape-worm, 
T.  confusa  of  which  only  segments  were  found  was  reported  by  Ward  from  Nebraska. 

Hymenolepis  nana  (Taenia  nana).— This  is  generally  known  as  the 
dwarf  tape-worm — -it  is  the  smallest  of  the  human  tape-worms.  It  is 
from  1/4  of  an  inch  to  1/2  inch  in  length,  and  is  less  than  1/25  of  an 
inch  in  breadth.  (zoX  i  m.m.) 

The  genus  Hymenolepis  has  lateral  genital  pores,  all  of  which  are  on  the  same 
side.  These  lateral  genital  pores  cannot  be  made  out  in  specimens  as  ordinarily 
examined.  The  head  has  four  suckers  and  a  rostellum,  which  is  usually  invaginated. 
The  rostellum  has  a  single  row  of  twenty-four  to  thirty  hooklets  encircling  it.  Of 
the  150  to  200  narrow  segments  the  terminal  ones  are  packed  with  eggs  which  in  the 
last  two  or  three  seem  to  fill  entirely  the  disintegrating  segments.  It  would  seem 
that  the  fully  mature  segments  disintegrate  and  in  this  way  the  eggs  are  set  free  in 
the  surrounding  intestinal  contents. 

The  worms  as  found  in  fresh  faeces  after  taeniacide  treatment  are  frequently  in 
an  advanced  state  of  disintegration  so  that  it  is  impossible  to  make  out  the  head  or 
hooklets. 

The  eggs  of  this  species  are  quite  characteristic,  there  being  two  distinct  mem- 
branes. The  inner  one  has  two  distinct  knobs,  from  which  thread-like  filaments 
proceed.  The  eggs  of  the  H.  diminuta  have  a  thicker,  striated,  outer  membrane 
and  there  are  no  filaments.  The  eggs  of  the  Dipylidium  caninum  are  similar,  but  are 
found  in  the  faeces  in  aggregations — several  eggs  in  a  packet. 

The  dwarf  tape-worm  has  been  found  to  be  the  most  common  tape- 
worm in  the  United  States.     Dr.  Stiles  found  it  in  about  5%  of  children 
in  a  Washington  orphanage. 
17 


258  FLAT   WORMS 

It  has  been  estimated  that  in  certain  parts  of  Italy  10%  of  the  children  may  be 
infected.  The  symptoms,  expecially  nervous  ones,  may  be  marked  in  this  infection. 
It  has  been  incriminated  as  a  cause  of  chyluria.  Although  very  small,  yet  the  num- 
ber of  parasites  may  be  very  great,  even  more  than  1000.  In  a  case  that  I  treated 
with  thymol  there  were  1500  worms  expelled.  A  form  found  in  rats,  which  may  be 
identical  with  H.  nana,  does  not  require  an  intermediate  host.  The  six-hooked 
embryo  bores  into  the  intestinal  villus  and  there  develops  a  Cercocystis  (larva  of 
small  dimensions  with  but  little  fluid).  When  fully  developed,  it  drops  into  the 
lumen  of  the  gut,  and  a  new  parasite  is  added  to  the  already  existing  number  of 
parasites.  This  explains  the  heavy  infection.  H.  diminuta  and  H.  lanceolata  have 
also  been  reported  for  man  a  few  times. 

H.  diminuta  is  much  larger  than  H.  nana,  being  about  10  inches  long.  The 
suckers  are  small  and  the  rostellum  insignificant  and  unarmed.  The  intermediate 
host  is  some  insect,  as  a  moth;  the  definitive,  the  rat.  As  man  is  not  liable  to  eat 
the  insect  hosts  the  infection  is  rare  in  man.  Twelve  cases  have  been  reported  for 
man  of  which  5  were  from  the  U.  S. 

H.  lanceolata  is  common  in  geese  and  ducks. 

Dipylidium  caninum  (Taenia  cucumerina)  (T.  flavopunctata) . — This  is  a  com- 
mon parasite  of  dogs  and  cats.  The  larval  stage  is  passed  in  lice  .and  fleas.  The 
cases  of  human  infection  have  been  principally  in  children,  probably  from  getting 
dog  lice  or  fleas  in  their  mouths.  The  number  of  infections  reported  for  man  is 
about  40  and  of  these  about  30  in  children.  The  head  has  four  suckers  and  a 
rostellum,  which  has  three  or  four  rows  of  encircling  hooklets.  The  segments  have 
the  shape  of  melon  seeds  and  have  bilateral  genital  pores. 

Davainea  madagascariensis. — This  tape-worm  has  been  found  in  Siam  and 
Mauritius.  It  is  about  10  inches  long.  The  head  has  four  suckers  and  a  rostellum 
with  ninety  hooklets.  The  suckers  have  rings  of  hooklets.  The  genital  pores  are 
unilateral.  The  cockroach  is  supposed  to  be  the  intermediate  host. 

There  have  been  about  10  cases  reported  (Madagascar,  Siam  and  British 
Guiana).  There  has  also  been  reported  a  D.  asiatica,  the  single  specimen,  however, 
lacking  a  head  so  that  the  exact  genus  is  doubtful.  It  has  been  reported  twice  in 
children  in  Breslau.  The  intermediate  host  is  thought  to  be  a  cyclops.  Garrison 
reported  cases  from  the  Philippines. 

DlBOTHRIOCEPHALID^E  INFECTIONS. 

Dibothriocephalus  latus  (Bothriocephalus  latus). — This  is  fre- 
quently termed  the  broad  Russian  tape-worm.  It  has  a  small  olive- 
shaped  head  with  two  deep  winding  suctorial  grooves  on  each  side ;  it  has 
neither  rostellum  nor  hooklets. 

The  segments  are  quite  broad.,  being  about  1/2  by  1/5  inch.  At  the  end  of  the 
strobila  they  are  more  nearly  square.  The  segments  are  very  numerous,  3000  or 
more.  The  fully  developed  worm  is  about  30  feet  long.  The  uterus  in  each  segment 
is  rosette-shaped  and  the  genital  pore  is  ventrally  situated.  The  eggs  of  this  species 
have  an  operculum  and  a  ciliated  embryo.  This  ciliated  embryo  swims  around  and 
either  enters  some  fish,  especially  pike,  directly  or  through  an  as  yet  unknown  inter- 


HYDATID    CYSTS  259 

mediary.  This  parasite  produces  an  intense  anaemia  similar  to  pernicious  anaemia. 
It  is  a  frequent  parasite  in  Switzerland,  Bavaria,  Japan,  Scandinavia,  and  Russia. 
Recently  several  cases  have  been  reported  from  our  Northwest,  and  some  of  the  fish 
of  the  waters  of  that  region  are  said  to  be  infected.  The  larva  is  a  pleroceroid  and 
is  about  i  inch  long.  It  is  said  that  salting,  smoking,  or  other  ordinary  methods  of 
preserving  fish  will  not  kill  it. 

A  tape-worm,  Diplogonopoms  grandis  has  been  reported  from  Japan.  In  this 
there  are  two  complete  sets  of  genital  organs  to  each  segment. 

SOMATIC  T^ENIASIS. 

While  rarely  we  may  have  the  larval  stage  of  T.  solium  present  in 
man,  and  while  certain  bothriocephalid  larvae  (Sparganum  mansoni 
and  Sparganum  proliferum)  infect  man,  yet  they  are  unimportant  as 


FIG.  68. — Daughter  cyst  from  FIG.  69. — A  group  of  daughter 

hydatid   cyst,  considerably   en-  cysts    from     hydatid     cysts, 

larged.     (Coplin.}  (Coplin.} 

compared  with  the  larval  stage  of  the  Taenia  echinococcus.  The 
adult  stage  of  this  parasite  is  passed  in  dogs.  It  is  one  of  the  smallest 
tape-worms  known,  being  only  about  1/6  inch  long.  It  has  a  head  with 
four  suckers  and  a  rostellum  encircled  with  hooks.  There  are  only  three 
to  four  segments.  The  larval  stage,  on  the  contrary,  gives  one  of  the 
largest  of  larval  cestodes.  In  man  it  may  reach  the  size  of  a  child's 
head.  The  larval  stage  is  also  found  in  hogs  and  sheep,  and  it  is  prob- 
able that  by  reason  of  the  dog's  eating  the  echinococcus  cyst  of  such 
animals  at  the  abattoir  we  owe  the  increase  in  this  serious  infection. 

Man  contracts  the  infection  from  association  with  dogs.  The  disease  is  peculiarly 
prevalent  in  Iceland.  As  stated  above,  the  adult  stage  is  passed  in  the  intestine  of 
the  dog.  Should  the  egg-bearing  segments  passed  by  the  dog  contaminate  the  hands 


260 


FLAT   WORMS 


of  man  and  a  single  egg  be  ingested,  we  may  have  hundreds  of  Taenia  larvae  produced. 
The  six-hooked  embryo,  leaving  its  shell,  bores  its  way  through  the  walls  of  the 
alimentary  tract  and  especially  seeks  the  liver,  just  as  the  embryo  of  T.  solium  seeks 
the  brain  and  eye. 

Griffith  notes  that  in  Australia  from  10  to  15%  of  hydatid  cysts 
occur  in  the  lungs.  The  cyst  wall  is  quite  thin  and  the  hydatid  cachexia 
seems  to  appear  earlier  in  the  lung  than  in  the  liver  cases. 

In  the  development  of  the  cyst,  after  the  embryo  has  come  to  rest  at  some  point 
in  the  liver,  we  have  formed  at  first  an  indistinctly  laminated  external  envelope 


s. 


FIG.  70. — Tape-worms,  i,  2,  and  3,  Head,  melon-shaped  segments  and  egg 
packet  of  Dipylidium  caninum;  4,  5,  6  and  10,  entire  worm  magnified,  head,  larval 
stage  in  intestinal  villus  and  ovum  of  Hymenolepis  nana;  7,  echinococcus  cyst;  A, 
mother  cyst;  D,  daughter  cyst;  E,  granddaughter  cyst;  C,  scolex  in  brood  capsule; 
B,  brood  capsule;  G,  parenchymatous  layer;  F,  laminated  layer;  8  and  9,  Taenia 
echinococcus;  9,  natural  size. 

with  coarsely  granular  fluid  contents.  Later  on  the  contents  become  transparent, 
and  two  distinct  layers  can  be  observed:  i.  The  external,  markedly  laminated  one, 
and  2.  the  internal  one,  made  up  of  small  cells  externally  and  large  cells  and  cal- 
careous corpuscles  internally.  This  internal  lining  membrane  is  known  as  the  paren- 
chymatous or  germinal  layer.  When  the  external  layer  is  incised  it  curls  up  by 
reason  of  its  elasticity.  This  is  characteristic  of  such  a  cyst.  In  addition,  we  have 
an  enveloping  connective-tissue  capsule  formed  by  the  surrounding  liver  substance. 
From  the  germinal  layer  arise  the  brood  capsules  and  the  scolices.  In  these  brood 


CESTODES 


261 


capsules  we  have  the  cellular  layer  external — just  the  reverse  of  the  mother  cyst. 
Scolices  may  develop  either  on  the  outside  or  inside  of  these  brood  capsules.  It  is 
interesting  to  note  that  one  onchosphere  may  develop  hundreds  of  scolices.  From 
the  parenchymatous  layer  of  the  mother  cyst,  daughter  cysts  are  formed;  these  have 
an  external  stratified  layer  and  an  internal  parenchymatous  one;  within  them  a  vary- 
ing number  of  scolices  may  develop.  From  these  daughter  cysts,  granddaughter 
cysts  may  arise — all  within  the  mother  cyst — and  hence  are  termed  endogenous. 

At  times  the  daughter  cysts  work  their  way  external  to  the  mother  cyst  and  pro- 
ceed to  develop  in  a  manner  similar  to  the  endogenous  formation.     The  exogenous 

Ova  or  the  Parasitic  Worms  or  Man 
CESTODA 

DRAWN  TO  SCALE  X    .    •      I  <S  O  O 


Tivniix 
solium 


n  Diplo^a^  Dibothrio^ 

cfonoporus    cephalus       nymenolepis  ntxna. 

^m^       3   f*r>cJ*^r*ln  1*>\tlI«S/  \  te.rtcrRon8Qm,fro«BSt.tlc»!9(O) 

/?%v    dr&nais.      iuius(*rten,o<m.«>«) 

If  'l)\m        O  (after  Ioo»».l$a6)  .  _,=-—         ¥-1  t  •     '• 

s^^\nymenolepi 

iw^«^%^r  ^x  \  diminuta 

TeemaXgp^  f%\  \^^^fn>.^^^ 

sadinata  . 

OM'f'i-^'iow)    T\-       i-  i 

Uipylidium 


num^mrs.^iooo) 

Cestode  sepments 

DRAWN     TO    SCALt    X    IO        W 

ggjj^^jgj      ^^MauMtTciy, 

Decvtvinea 


Eni  njL,    SSL-  *"!fc 

solium  eaninum     Dibothriocephalus  latus  — s 

m*™/""  (/.S.A'tu-atMedicaJ School. 


FIG.  71. — Cestode  ova. 

development  is  rare  in  man,  but  common  in  hogs.  Hydatids  containing  no  scolices 
are  called  sterile.  These  cysts  may  be  as  large  as  a  child's  head,  but  are  usually 
smaller.  The  fluid  of  these  cysts  contains  about  i%  of  NaCl,  also  a  trace  of  sugar; 
in  addition  there  is  a  toxin  which  produces  urticaria  and  acts  as  a  cardiac  depressant. 
If  any  quantity  should  escape  into  the  peritoneal  cavity  at  operation,  it  may  cause 
death.  Hydatids  develop  very  slowly,  and  the  duration  of  the  disease  is  usually 
from  two  to  eight  years. 

Echinococcus  multilocularis  is  possibly  due  to  a  species  different  from  T.  echino- 
coccus.     In  this  we  have  a  honeycomb  arrangement  with  cavities  filled  with  a  gela- 


262  FLAT    WORMS 

tinous  material.     The  majority  of  these  cysts  are  without  scolices.     This  form  of 
hydatid  is  very  fatal. 

Sparganum  mansoni  (Bothriocephalus  liguloides) . — This  is  a  larval  bothrio- 
cephalid  which  is  about  5  to  10  inches  long  and  has  been  reported  ten  times  in  Japan. 
It  has  been  found  in  various  parts  of  the  body,  as  in  pleural  cavity,  tissues  about 
kidney,  and  in  abscess  of  the  thigh.  They  have  been  found  in  the  urethra  and  under 
the  conjunctiva.  They  resemble  ribbon-like  strings  of  fat. 

Sparganum  prolifer  (Plerocercoides  prolifer). — This  has  been  reported  from 
Japan  as  a  larval  form  in  the  subcutaneous  tissue.  Stiles  has  found  these  larval 
forms  in  skin  lesions  in  Florida.  They  show  themselves  as  bizarre  grub-like  forms. 
They  reproduce  by  budding. 


CHAPTER  XVIII. 


THE  ROUND  WORMS. 


CLASSIFICATION  or  THE  NEMATHELMINTHES  (ROUND  WORMS  ) . 


Class. 


Nematoda 


Family. 
Angiostomidae 


Filariidae 


Genus. 


Species. 


Trichotrachelidae 


Strongylidae 


Ascaridae 


Strongyloides 

S. 

stercoralis. 

Dracunculus 

D. 

medinensis 

F. 

bancrofti 

F. 

loa 

F. 

perstans 

Filaria 

F. 

demarquayi 

F. 

ozzardi 

F. 

philippinensis 

IF. 

volvulus 

Trichuris 

T. 

trichiura 

L  Trichinella 

T. 

spiralis 

Eustrongylus 

E. 

gigas 

Strongylus 

S. 

apri 

Trichostrongylus 

T. 

instabilis 

Triodontophorus 

T. 

deminutus 

CEsophagostoma 

0. 

brumpti 

Phy  salop  tera 

P. 

caucasica 

Ancylostoma 

A 

duodenale 

Necator 

N.  americanus 

Ascaris                        < 

f  A. 

A 

lumbricoides 

t  A. 

canis 

[  Oxyuris 

O. 

vermicularis 

Gigantorhynchus 

G.  gigas 

{Hirudo 

H 

medicinalis 

Limnatis 

L.  nilotica 

Hasmadipsa 

H.  ceylonica 

Acanthocephala 
Hirudinea 


NOTE. — The  Strongyloides  stercoralis  was  formerly  described  under  two  desig- 
nations: (i)  Anguillula  intestinalis,  a  parasitic  generation  and  (2)  Anguillula  ster- 
coralis, a  free  living  generation. 

ROUND  WORMS  OR  NEMATODES. 

All  nematodes  are  covered  by  a  cuticle  which  varies  in  thickness, 
and  is  frequently  ringed.  The  cuticle  is  moulted  three  or  four  times. 
The  cuticle  is  formed  by  the  underlying  ectoderm  which  is,  as  a  rule, 

263 


264  THE   ROUND    WORM 

markedly  developed  in  four  ridges  which  divide  the  body  into  quadrants. 
Within  the  ectoderm  is  the  body  cavity,  a  space  in  which  the  reproduc- 
tive organs  lie  in  a  clear  fluid.  The  excretory  system  usually  consists 
of  two  tubes  which  discharge  near  the  head. 

While  the  alimentary  canal  is  more  or  less  tube  like  in  appearance  it  shows  near 
the  mouth  a  muscular  oesophagus  with  a  bulb-like  expansion  at  the  commencement 
of  the  remainder  of  the  intestinal  tract. 

The  testis  and  ovary  are  generally  tube  like.  The  sexes  are,  as  a  rule,  separate. 
The  male  can  usually  be  recognized  by  its  smaller  size,  its  curved  or  curled  posterior 
end,  and  at  times  exhibiting  an  umbrella-like  expansion — the  copulatory  bursa. 
The  spicules,  chitinous  copulatory  structures,  may  be  observed  drawn  up  in  the  worm 
or  projected  out  of  the  cloaca.  The  genital  opening  of  the  female  is  ventral  and 
usually  about  the  mid-point;  that  of  the  male  is  close  to  the  anus. 

Certain  papillae  in  the  region  of  the  anus  are  valuable  in  differentiation.  As  a 
rule  nematodes  develop  in  damp  earth  from  the  eggs  as  rhabditiform  larvae.  Very 
few  nematodes  are  viviparous  (Filaria,  Trichinella). 

The  families  Gnathostomidae  and  Anguillulidae  are  of  very  little 
importance  in  human  parasitology.  Gnathostoma  siamense  was  once 
found  in  a  breast  tumor  and  Rhabditis  pellio  once  in  the  urine. 

Anguillula  aceti,  the  vinegar  eel,  has  been  reported  from  the  genito-urinary  tract 
several  times.  Such  cases  can  be  explained  by  the  prior  contamination  of  the  urine 
bottle  or  by  the  use  on  the  part  of  the  patient  of  a  vinegar  vaginal  douche.  The 
genera  Rhabditis  and  Anguillula  belong  to  the  family  Anguillulidae. 

A  case  of  infection  with  a  small  nematode  found  in  the  papules  of  a  skin  infection, 
in  a  French  boy  is  recorded  as  due  to  Rhabditis  niellyi.  The  present  view  is  that 
the  parasites  were  embryos  of  A.  duodenale,  boring  into  the  skin. 

ANGIOSTOMHXE. 

In  this  family  we  have  heterogenesis. 

Strongyloides  stercoralis. — This  parasite  was  formerly  thought 
to  be  the  cause  of  Cochin-China  diarrhoea.  It  presents  two  genera- 
tions: i.  Parasitical  or  intestinal  form.  2.  The  free  living  or  faecal 
form. 

i.  The  intestinal  form  (also  known  as  Anguillula  intestinalis)  is  represented  only 
by  females.  These  are  about  1/12  of  an  inch  (2  mm.)  long  and  reproduce  partheno- 
genetically.  They  have  a  pointed,  four-lipped  mouth,  and  a  filariform  oesophagus 
which  extends  along  the  anterior  fourth  of  the  body.  The  anus  is  situated  near  the 
sharpened  posterior  end,  the  vulva  about  the  lower  third  of  the  body.  The  uterus 
contains  a  row  of  8  to  10  elliptical  eggs  which  stand  out  prominently  in  the  posterior 
part  of  the  body  by  reason  of  being  almost  as  wide  as  the  parent  worm.  They 
usually  live  deep  in  the  mucosa  and  the  embryos  emerge  from  the  ova  laid  in  the 


FILARIASIS  265 

mucosa.  The  embryos  escape  from  the  eggs  while  still  in  the  intestine,  so  that  in 
the  faeces  we  only  find  actively  motile  embryos.  The  eggs,  which  are  strung  out 
in  a  chain,  never  appear  in  the  faeces  except  during  purgation.  As  they  greatly 
resemble  hookworm  eggs,  this  is  a  point  of  great  practical  importance.  In  fresh 
faeces  we  find  hookworm  eggs  and  Strongyloides  embryos.  The  embryos  are  rather 
common  in  stools  in  the  tropics.  These  embryos  have  pointed  tails  and  are  about 
250  X 13/4.  They  have  a  double  cesophageal  bulb.  They  are  about  250^  when  they 
first  emerge  but  may  grow  until  they  will  approximate  500/4  in  the  faeces.  If  the 
temperature  is  low,  these  rhabditiform  embryos  develop  into  filariform  embryos, 
which  being  ingested  form  the  infecting  stage.  It  has  been  demonstrated  that 
infection  of  man  may  also  take  place  through  the  skin.  If  the  temperature  is  warm, 
25°  to  35°  C.,  these  embryos  develop  into: 

2.  The  free  living  form,  Anguillula  stercoralis.  In  this  we  have  males  and 
females,  with  double  oesophageal  bulbs,  the  male  about  1/30  of  an  inch  (3/4  mm.)  long 
with  an  incurved  tail  and  2  spicules  and  the  female  about  1/25  inch  (i  mm.)  long 
with  an  attenuated  tail;  these  copulate  and  we  have  produced  rhabditiform  larvae, 
which  later  change  to  filariform  ones.  At  this  time  the  length  is  about  550  microns. 
These,  being  ingested,  start  up  the  parasitical  generation.  If  these  do  not  reach  the 
intestine  they  die  out. 


FILARIID.E. 

This  family  is  of  the  greatest  importance  to  man.  It  is  also  one 
about  which  much  confusion  exists  as  to  the  adult  type;  hence  anyone 
finding  adult  filariae  should  fix  them  in  hot  5%  glycerine  alcohol 
(alcohol  70%),  and  subsequently  mount  in  glycerine  gelatin.  Formalin 
is  not  to  be  used,  other  than  for  a  very  brief  period  (2  to  6  hours)  and 
then  followed  by  the  lacto-phenol  method. 

These  worms  are  most  likely  to  be  seen  as  writhing  thread-like  worms,  especially 
in  the  lymphatic  glands  and  connective  tissue,  and  about  body  cavities.  They  have 
a  lipped  or  simple  mouth  and  a  filariform  oesophagus.  The  male  has  an  incurved 
tail  with-preanal  and  postanal  papillae  which  may  be  even  corkscrew-like  as  in  F. 
immitis.  The  spicules  are  unequal  or  there  may  be  but  one.  The  female  is  ovovivi- 
parous,  the  vulva  is  at  the  anterior  end  and  the  uterus  usually  double. 

Dracunculus  medinensis  (Filaria  medinensis).— The  Guinea  or 
Medina  worm,  of  which  until  recently  only  the  female  was  known,  is 
of  great  importance  in  parts  of  India,  Africa,  and  Arabia.  The  female 
is  a  thread-like  worm,  about  20  to  30  inches  long.  The  habitat  is  the 
subcutaneous  and  intermuscular  connective  tissue,  especially  of  the 
lower  extremity.  It  develops  without  symptoms.  Finally  a  blister- 
like  area  appears  on  the  surface  of  the  leg,  particularly  about  ankle- 
joint,  which  soon  forms  a  painful  ulcer.  From  this  opening  the 


266 


THE   ROUND    WORMS 


anterior  end  of  the  worm  projects  to  pour  forth  its  striated  embryos 
upon  contact  with  water. 

The  mouth  is  terminal  and  the  body  uniformly  cylindrical.  The  uterus  is  a  con- 
tinuous tube  filled  with  sharp-tailed,  transversely  striated  embryos,  650X17^,  and 
constitutes  the  greater  part  of  the  body,  the  alimentary  canal  being  pressed  to  one 
side.  The  genital  organs  probably  discharge  through  the  oesophagus.  The  body 
when  being  extracted  is  rather  transparent.  The  tip  of  the  tail  is  bent,  forming  a 
sort  of  anchoring  hook.  Recently  Leiper  fed  monkeys  on  bananas  containing  in- 


7-08. 


FIG.  72. — Round  worms  (Filariidae).  i.  Hooked  posterior  extremity  and  ante- 
rior extremity  of  Dracunculus  medinensis;  2,  cross  section  of  uterus  filled  with  embryos, 
D.  medinensis;  3  and  4,  free  embryo  and  embryos  of  D.  medinensis  in  intermediate 
host  (Cyclops);  5,  natural  size  of  female  Filaria  bancrofti;  6,  embryo  of  F.  bancrofti 
in  blood; 1 7,  tail  of  male  F.  bancrofti;  8,  male  and  female  of  F.  loa  (natural  size); 
9,  tuberculated  integument  and  posterior  end  of  male  F.  loa;  10,  posterior  end  of  male 
F.  perstans;  n,  male  of  F.  bancrofti  (natural  size);  12,  blunt-tailed  embryo  of  F.  per- 
stans;  13,  sharp-tailed  embryo  of  F.  demarquayi. 

fected  Cyclops,  and  at  the  autopsy  six  months  later  obtained  both  male  and  female 
forms. 

As  regards  the  life  history,  Fedschenko,  in  1870,  showed  that  the  embryos  when 
liberated  swam  around  in  water  and  finally  entered  the  bodies  of  species  of  the  genus 
Cyclops.  The  female  tends  to  come  to  the  surface  in  the  lower  extremities,  and 
experiments  show  that  if  on  the  blister-like  points  of  emergence  some  water  be 
squeezed  out  from  a  sponge,  the  uterus  will  eject  a  milky-looking  fluid  containing 


FILARIASIS  267 

myriads  of  embryos.  This  would  indicate  that  the  worm  selects  the  lower  extremity 
so  that  the  embryos  may  gain  access  to  the  Cyclops  when  the  host  is  wading  through 
the  water. 

Leiper  showed  that  a  strength  of  HC1  equal  to  that  of  gastric  juice  killed  the 
Cyclops,  but  made  the  Dracunculus  embryos  very  active.  From  this  he  judged  that 
infection  must  probably  take  place  from  drinking  water  containing  infected  Cyclops. 
The  suggestion  of  Leiper  that  wells  harboring  Cyclops  be  treated  with  steam,  intro- 
duced by  a  pipe,  seems  to  be  valuable.  The  disease  is  known  as  "Dracontiasis." 

Filaria  loa  (Filaria  oculi). — This  is  a  thread-like  worm  of  West 
Africa  about  i  to  2  inches  long.  The  cuticle  is  characterized  by 
distinct  wart-like  structures. 

The  anterior  extremity  is  like  a  truncated  cone  with  two  papillae  at  the  base  of 
the  cone.  The  wart-like  cuticular  protuberances  or  bosses  are  about  12  to  15 
microns  in  height.  The  females  are  2  to  3  inches  (50  to  70  mm.)  long  and  about  1/2 
mm.  broad. 

The  males  are  smaller  than  the  females  and  have  three  preanal  papillae  and  two 
postanal  ones.  There  are  two  short  unequal  spicules.  The  life  history  is  not  satis- 
factorily established.  The  young  are  born  ovoviviparously,  and  it  has  been  sug- 
gested that  the  localized  oedemas,  known  as  Calabar  swelling,  may  be  due  to  the 
irritation  produced  by  these  eggs.  These  swellings  are  of  hen's  egg  size,  painless, 
do  not  pit  on  pressure  and  last  about  three  days.  They  occur  especially  on  the  hands 
and  arms.  The  embryos  almost  exactly  resemble  those  of  F.  bancrofti.  They  have 
a  diurnal  periodicity,  however,  appearing  in  the  blood  about  8  A.  M.,  increasing  to 
noon  and  disappearing  about  9  p.  M.  The  adult  worms  have  a  tendency  to 
wander  about  in  the  subcutaneous  connective  tissue,  especially  about  the  region  of  the 
orbit  or  even  under  the  conjunctiva. 

Adult  worms  of  F.  loa  have  been  found  and  extracted,  with  an  absence  of  the 
filarial  embryos  in  the  peripheral  circulation  of  the  patient.  Leiper  has  just  noted 
two  species  of  Chrysops  as  intermediate  hosts,  the  embryos  developing  in  the 
salivary  glands. 

Filaria  bancrofti  (Filaria  sanguinis  hominis). — -This  is  the  most 
important  of  the  filarial  worms.  It  is  a  common  infection  in  South 
China,  India,  the  West  Indies,  and  in  the  Pacific  Islands,  especially 
Samoa. 

In  medical  books  the  embryos  have  been  designated  Filaria  sanguinis  hominis. 
This  species  is  the  cause  of  the  common  manifestations  of  filariasis,  such  as  elephanti- 
asis, varicose  groin  glands,  chyluria,  lymph  scrotum,  etc. 

Filarial  diseases  are  prone  to  lymphangitis  attacks.  Thus  in  lymph  scrotum  an 
erysipelatoid  condition  of  the  scrotum  with  high  fever  and  chills  may  result.  This 
condition  is  at  times  mistaken  for  malaria.  Varicose  groin  glands  may  be  mistaken 
for  hernia.  In  the  Philippines  very  few  symptoms  are  noted  in  those  affected  with 
filariasis.  Occasionally  chylocele  or  chyluria  is  reported. 

F.  bancrofti  lives  in  lymphatics  of  trunk  and  extremities.  At  times  the  fine 
white  thread-like  worjms  may  be  seen  as  writhing  coils  in  lymphatic  glands. 


268  THE    ROUND    WORMS 

The  sexes  are  usually  found  together.  The  females  are  about  3  inches  long 
and  the  males  less  than  2  inches.  The  tails  of  both  sexes  are  incurved,  but  that 
of  the  male  is  more  so.  The  head  is  club-shaped.  The  vulva  opens  1.2  mm.  from 
the  anterior  end.  There  are  2  uterine  tubules.  The  sheathed  embryos  are  supposed 
to  be  born  viviparously  and  Manson  supposes  that  as  a  result  of  injury  to  the  parent 
worm  and  resulting  extrusion  of  eggs,  the  blocking  of  lymph  channels  occurs. 

A  very  interesting  fact  is  that  people  with  elephantiasis  fail  to  show  larvae  in  the 
peripheral  circulation.  Manson  considers  that  it  is  due  to  the  blocking  of  the  lymph 
channels. 

These  embryos  show  a  nocturnal  periodicity.  During  the  day  they 
remain  in  the  lungs,  and  larger  arteries. 

If  the  patient  sleeps  in  the  day  time  and  is  active  at  night  the  nocturnal  perio- 
dicity or  presence  of  embryos  in  peripheral  circulation  is  inverted.  In  the  case  of 
F.  loa,  however,  a  change  of  habits  does  not  change  the  periodicity  of  the  filarial 
embryos,  they  continue  to  appear  in  the  peripheral  circulation  by  day  even  if  the 
patient  sleeps  at  that  time. 

The  disease  is  transmitted  especially  by  Culex  fatigans.  The  sheathed  embryos, 
getting  into  stomach  of  mosquito,  wriggle  out  of  the  sheath,  they  then  bore  their 
way  through  walls  of  stomach  and  enter  into  a  sort  of  passive  stage,  during  which 
further  development  takes  place.  They  finally  become  distributed  in  the  muscles 
of  the  thorax  and  make  their  way  along  the  fleshy  labium,  to  enter  the  wound  in 
a  person  bitten  by  a  mosquito,  by  way  of  Button's  membrane.  This  takes  about 
twenty  days  at  which  time  the  larvae  are  about  1/16  inch  long  and  have  an  alimentary 
canal. 

Filaria  perstans. — The  adults  are  found  in  connective  tissue  and  deeper  fat, 
especially  about  the  mesentery  and  abdominal  aorta. 

The  female  is  about  3  inches  (75  mm.)  long;  the  male  is  rarely  found  and  is  less 
than  2  inches  long.  These  worms  are  characterized  by  incurved  tails,  the  extremity 
of  which  has  two  triangular  appendages  giving  a  bifid  appearance.  The  embryos 
do  not  possess  a  sheath  and  have  a  blunt  tail.  The  life  history  is  unknown.  Both 
mosquito  and  tick  have  been  incriminated.  The  embryos  are  always  present  in 
the  peripheral  circulation — hence  perstans.  There  does  not  seem  to  be  any  symp- 
tomatology. 

It  is  of  historical  interest  that  F.  perstans  was  once  considered  the  cause  of 
sleeping  sickness. 

Filaria  volvulus. — This  is  a  rather  common  parasite  of  Central  Africa.  The 
male  is  about  11/2  inches  (35  mm.)  and  the  female  about  5  inches  long.  The 
females  are  so  interlaced  in  the  fibro-cystic  swellings  that  it  is  difficult  to  determine 
their  length.  The  tumors  start  from  the  presence  of  a  worm  in  a  lymphatic.  The 
tumors  are  easily  enucleated.  Adults  are  striated.  They  are  found  in  cystic  tumors, 
especially  about  the  axilla  and  popliteal  space.  The  cystic  contents  contain  abundant 
sheathless  larvae  about  300/4  long;  they  are  not  found  in  the  peripheral  circulation. 
Life  history  unknown,  although  it  has  been  suggested  that  a  species  of  Glossina 
may  be  concerned. 

Filaria  demarquayi. — The  habitat  of  this  filarial  worm  is  the  West  Indies. 
The  embryo  has  no  sheath  and  has  a  sharp  tail.  Other  filarial  species  which  have 


WHIP   WORMS  269 

been  reported  are  F.  magalhaesi,  F.  ozzardi,  F.  volvulus,  F.  powelli,  and  F.  philip- 
pinensis.  A  species  called  F.  gigas  is  now  considered  to  have  been  only  the  hair 
of  the  leg  of  a  fly.  The  embryos  have  usually  been  given  such  names  as  F.  nocturna, 
F.  diurna,  etc.  Of  course  the  embryos  and  the  parent  should  have  the  same  name. 
It  has  been  proposed  to  designate  these  embryos  the  same  as  the  parent,  but  with 
the  use  of  the  term  Microfilaria  instead  of  Filaria. 

The  points  usually  noted  in  the  description  of  filarial  embryos  are: 

1 .  Presence  or  absence  of  periodicity  of  embryos  in  peripheral 
circulation. 

2.  Presence  or  absence  of  a  sac  sheath  around  the  embryo. 

3.  Accurate  measurements. 

4.  Shape  and  description  of  head  and  tail  ends. 

5.  Character  of  movement. 

6.  Location  of  V  spot  and  break  in  cell  column  in  stained 
specimens. 

KEY  TO  FILARIAL  LARVAE. 

A .  Sheath  present. 

1.  No  periodicity. 

F.  philippinensis.  Tightly-fitting  sheath;  not  flattened  out  beyond  extremi- 
ties. Tail  is  pointed  and  abruptly  attenuated.  Lashing  progression  move- 
ment. 320X6.5^. 

2.  Periodicity  exhibited. 

a.  Nocturnal  periodicity. 

F.  bancrofti  (F.  nocturna).     Pointed  tail;  loose  sheath;    lashing  movement. 
300X7.5,".     V  spot  QOjW  from  head;  break  in  cells  50^  from  head. 

b.  Diurnal  periodicity. 

F.  loa  (F.  diurna).     Pointed  tail;  loose  sheath;  245  by  7  microns.     V  spot 
60  to  70  microns  from  head,  break  in  cells  40  microns  from  head. 

B.  Absence  of  sheath.     None  of  these  exhibit  a  periodicity,  being  continuously  present. 

1.  Blunt  tail — F.  perstans.     200X4.5^- 

2.  Sharply-pointed  tail: 

a.  F.  demarquayi.     2ioX5/*. 

b.  F.  ozzardi.     215X5,". 

NOTE. — A  filarial  embryo,  F.  powelli,  reported  once.  It  has  a  sheath,  nocturnal 
periodicity,  and  is  about  i^oXs^1- 

TRICHOTRACHELID^E. 

These  have  a  long  thin  neck  and  a  thicker  terminal  portion.  The 
oesophagus  is  of  the  single  row  of  cells  type.  The  anus  is  terminal; 
there  is  only  one  ovary. 

Trichuris  trichiura  (Trichocephalus  dispar). — This  is  usually  called 


270 


THE    ROUND    WORMS 


the  whip-worm — 'the  thickened  body  representing  the  handle  and  the 
narrow  neck  the  lash.  It  is  one  of  the  most  common  parasites  in  both 
temperate  and  tropical  climates. 

The  egg  is  very  characteristic  in  having  an  oval  shape  with  knobs  at  either  extrem- 
ity. It  resembles  a  platter  with  handles.  The  male  is  almost  2  inches  long,  and 
has  the  terminal  portion  curled  up  in  a  spiral.  It  has  a  single  terminal  spicule. 

The  female  is  a  little  longer  than  the  male,  and  has  the  terminal  part  in  the  shape 
of  a  comma  instead  of  being  coiled.  The  neck  only  contains  the  oesophagus  which 


FIG.  73. — Round  worms,  i,  Encysted  embryo  of  Trichinella  spiralis;  2,  male 
and  female  of  T.  spiralis;  3,  male  and  female  of  Trichocephalus  trichiurus;  4,  egg 
of  T.  trichiurus;  5  and  6,  head  and  male  and  female  of  Ascaris  canis;  7,  8,  and  9,  head, 
egg  and  male  and  female  of  Oxyuris  vermicularis;  10,  n  and  12,  head,  egg  and  tail  of 
Ascaris  lumbricoides;  13  and  14,  head  and  egg  of  Echinorhynchus  gigas;  15,  16  and 
17,  parthenogenetic  female  and  rhabditif orm  and  mariform  embryos  of  Strongyloides 
stercoralis. 

is  contained  in  a  groove  in  large  cells  which  form  a  single  row  like  a  string  of  pearls. 
These  cells  play  a  digestion  role.  The  vulva  opens  at  the  upper  end  of  the  thickened 
terminal  end  which  contains  an  intestine  lying  between  the  ovary  and  uterus. 
The  great  powers  of  resistance  of  the  ova  may  account  for  their  general  distribution; 
they  may  live  for  months  under  conditions  of  freezing  and  so  forth.  There  is  no 
intermediate  host.  The  worm  arrives  at  sexual  maturity  in  about  one  month  after 
ingestion.  The  whip-worm  prefers  the  caecum,  but  also  lives  in  the  lower  end  of  the 
ileum  and  the  appendix. 


TRICHINOSIS 


271 


The  neck  burrows  into  the  mucosa,  and  much  importance  has  been  attributed 
by  the  French  to  the  possibility  of  this  paving  a  way  for  the  entrance  of  pathogenic 
bacteria.  They  do  not  seem  to  produce  serious  symptoms. 

Trichinella  spiralis  (Trichina  spiralis). — -The  cause  of  trichinosis  is 
usually  termed  Trichina  spiralis  in  medical  works. 

The  adults  live  in  the  duodenum  and  jejunum;  the  males  are  about  1/16  of  an 
inch  (1.5  mm.)  long  with  two  tongue-like  caudal  appendages  and  without  a  spicule. 
These  two  lateral  projections  enable  the  male  to  hold  the  female  in  copulation — 
the  cloaca  being  evaginated  to  act  as  a  penis. 

The  females  are  about  1/7  of  an  inch  (0.3  to  0.4  mm.)  long.  The  female  gives 
off  embryos  from  the  vulva  which  is  near  the  mouth  end  (viviparous). 

These  parasites  can  be  seen  with  an  ordinary  magnifying  glass.     With  higher 


FIG.   74. — Trichina  spiralis  (Ziegler). 

powers  the  oesophagus  has  the  appearance  of  a  serrated  line  instead  of  an  cesophageal 
bulb.  The  male  is  about  40^  broad  and  has  a  prominent  testicular  enlargement 
filling  the  posterior  extremity.  The  female  is  about  6o/x  broad  and  has  a  rounded 
posterior  extremity  with  a  prominent  slit-like  cloaca.  It  is  in  this  posterior  extremity 
that  the  female  increases  in  size  as  she  becomes  filled  with  eggs.  The  vulva  is  in  the 
anterior  third.  After  fertilization  of  the  females  the  males  die,  and  the  females 
bore  into  the  intestinal  mucosa  and  begin  to  produce  embryos  to  the  number  of 
more  than  1000  each.  These  gain  access  to  the  lymph  channels  and  are  distributed 
by  the  blood  stream  to  the  striated  muscles.  Embryos  reaching  other  tissues  fail  to 
develop. 

It  is  about  ten  days  before  they  reach  the  muscle.  In  the  muscle  they  become 
encysted  as  the  oval  lemon-shaped  areas  containing  coiled-up  embryos  that  every- 
one is  familiar  with.  These  oval  areas  are  about  450X250;*  and  have  a  chitinous 
capsule. 

The  encysted  trichinae  are  found  chiefly  in  the  muscle  fibers  of  the  tongue  and 


272  THE    ROUND    WORMS 

diaphragm  and  may  remain  alive  as  long  as  ten  to  twenty  years;  finally,  however, 
the  cyst  undergoes  calcareous  infiltration  and  the  embryo  dies. 

When  uncoiled  the  embryo  is  about  i  mm.  long  with  the  mouth  at  the  attenuated 
end.  Among  cannibals  it  would  be  easy  to  keep  the  cycle  going  by  eating  improperly 
cooked  or  raw  human  meat,  the  parasite  being  thus  transmitted. 

As  this  would  not  explain  the  transmission  among  civilized  men,  the 
following  is  the  life  history:  Man  obtains  his  infection  from  eating  raw 
pork,  the  embryos  encysted  in  the  muscle  of  the  hog  being  liberated  in 
the  stomach,  and  the  males  and  females  developing  in  the  intestine  as 
above  described.  The  hog  may  gain  his  infection  by  eating  the  meat 
of  other  hogs  or  rats.  These  rats  eat  scraps  of  pork  at  slaughter  houses 
and  become  infected.  Being  cannibals,  rats  when  once  infected,  con- 
tinue to  propagate  the  infection.  In  man,  during  the  first  two  or  three 
days  while  the  adults  are  breeding  in  the  intestine,  we  have  gastroin- 
testinal symptoms. 

It  is  during  this  period  or  at  any  rate  before  the  fifth  day  that 
purging  may  be  of  benefit.  About  ten  to  twenty  days  after  infection 
the  embryos  begin  to  wander  and  we  have  the  acute  muscle  pains. 
In  the  diagnosis  we  should  try  to  obtain  specimens  of  the  pork  which 
has  caused  the  trouble  in  order  to  examine  for  encysted  trichinae,  or 
to  feed  to  white  rats  or  rabbits,  subsequently  examining  the  diaphragm 
of  these  animals  for  encysted  trichinae  or  the  intestine  for  adult  trichinae. 
Excision  of  a  small  piece  of  the  deltoid  of  man  may  confirm  the 
diagnosis.  The  best  method  is  to  take  blood  in  3%  acetic  acid, 
centrifuge,  and  examine  for  larvae. 

During  the  diarrhceal  stage  we  may  examine  the  stools  for  adult  worms,  in 
particular  dead  males  or  possibly  actively  motile  embryos — these  latter  are  about 
90X6^. 

Always  examine  the  blood  for  eosinophilia. 

It  is  well  to  remember  that  the  parts  of  meat  which  trichinae  prefer  (muscle  of 
diaphragm,  of  neck,  etc.)  are  often  used  in  sausage.  Unfortunately  it  is  almost 
impossible  to  detect  the  embryos  in  sausage  meat. 

STRONGYLID.E. 

In  this  family  the  male  has  a  caudal  bursa,  a  prehensile  sort  of  ex- 
pansion at  the  posterior  end  for  copulatory  purposes. 

The  mouth  is  usually  provided  with  six  papillae  and  at  times  with  a 
chitinous  armature.  Those  without  the  chitinous  armature  are  in- 
cluded in  the  subfamily  Strongylinae  (Strongylus,  Trichostrongylus) 


STRONG  YLIDJ2  273 

while  those  having  an  armed  mouth  are  in  the  subfamily  Sclerosto- 
minae  (Ancylostoma,  Necator,  Triodontophorus,  (Esophagostoma, 
Physaloptera). 

Eustrongylus  gigas  (Strongylus  renalis). — -This  is  the  largest  round 
worm  infecting  man;  it  is  usually  found  in  the  pelvis  of  the  kidney 
(giant  strongyle). 

Two  or  more  worms  may  so  distend  the  kidney  as  to  convert  it  into  a  mere  shell. 
Pain,  haematuria  and  other  symptoms  of  pyuria,  together  with  the  finding  of  the  eggs, 
make  the  diagnosis.  There  seem  to  be  seven  authentic  and  eight  doubtful  cases 
of  infection  in  man. 

The  females  are  about  40  inches  (i  m.)  long  and  about  1/3  of  an  inch  (8  mm.) 
in  breadth  while  the  male  is  about  10  inches  (25  cm.)  long. 

The  collar-like  copulatory  bursa  of  the  male  distinguishes  it  from  Ascaris  as 
does  also  the  dark  red  color.  The  source  of  infection  is  unknown  but  it  has  been 
suggested  that  the  larval  stage  may  exist  in  fish. 

Many  of  the  reported  cases  were  simply  fibrinous  clots  from  ureters  or  wandering 
round  worms. 

The  very  characteristic  ova,  with  gouged-out  oval  depressions,  may  be  found 
in  the  urine,  and  are  diagnostically  confirmatory. 

Strongylus  apri. — This  nematode  is  common  in  the  lungs  of  hogs,  producing 
a  bronchitis  in  young  animals  but  apparently  harmless  for  adult  ones.  It  has  been 
reported  once  from  the  lungs  of  a  six-year-old  boy.  The  male  is  about  i  inch  (25 
mm.)  long  with  two  long  spicules.  The  female  is  about  2  inches  long  and  has  a 
sharply  hooked  posterior  extremity  with  the  vulva  just  beyond  the  bend.  The  mouth 
has  six  lips.  The  eggs  contain  embryos  when  laid. 

Trichostrongylus  instabilis. — This  is  a  small  strongyle  formerly  known  as  Stron- 
gylus subtilis.  The  male  is  about  1/6  of  an  inch  (4  mm.)  long,  and  the  female  about 
1/4  of  an  inch  (6  mm.).  Anteriorly  it  tapers  to  a  pointed  head  end  which  is  only 
about  one-tenth  the  thickness  of  the  posterior  extremity.  The  male  has  a  bursa 
and  two  prominent  equal  spicules.  It  has  been  found  in  the  upper  part  of  the  small 
intestine  of  inhabitants  of  Egypt  and  Japan.  It  does  not  appear  to  produce  symp- 
toms. Ova  like  hookworm  ones  (63  X  4 1  ft)  • 

Triodontophorus  deminutus. — This  is  a  small  round  worm  with  three  forked 
teeth  taking  origin  from  the  pharyngeal  lobes.  The  collar-like  mouth  orifice  is 
made  up  of  22  rounded  plates  just  inside  the  round  mouth  opening.  They  are  less 
than  1/2  inch  long  and  have  once  been  found  in  the  intestinal  canal. 

(Esophagostoma  brumpti. — Six  young  females  were  found  in  a  cyst  of  the  colon 
in  an  African  negro.  They  were  about  1/3  inch  (8  mm.)  long.  The  anterior  end 
presents  an  ovoid  protuberance  with  a  second  cuticular  inflation  just  below  it.  The 
buccal  capsule  is  very  shallow  and  surrounded  by  about  a  dozen  chitinous  plates. 
The  mouth  has  six  papillae. 

This  species  has  recently  been  reported  by  Thomas  in  a  native  of  Brazil. 

Physaloptera  caucasica. — Mouth  with  two  equal  laterally  placed  lips,  each  hav- 
ing three  papillae  and  three  teeth.  The  male  has  a  lancet-shaped  posterior  extremity 
and  is  about  1/2  inch  long  (14  mm.  by  0.71  mm.).  Female  is  about  i  inch  long 
18 


274  THE    ROUND    WORMS 

(27  mm.)  with  a  rounded  tail  end.  Found  only  once  in  the  alimentary  canal  of  a 
native  in  the  Caucasus.  Leiper  has  recently  reported  a  species  P.  mordens  from 
Uganda,  one  case. 

Ancylostoma  duodenale  (Dochmius  duodenalis.) — The  hookworm, 
so  called  from  the  hook-like  appearance  of  the  ribs  of  the  copulatory 
bursa  or  from  the  hook-like  projection  of  the  head  dorsally,  is  probably 
the  most  important  of  the  parasitic  worms.  This  species  in  Europe 
and  Africa  and  the  Necator  americanus  in  the  New  World  cause  an 
immense  amount  of  invaliding.  The  Egyptian  anaemia  and  the  Porto 
Rican  anaemia  are  caused  by  this  parasite. 

Goeze  found  a  hookworm  in  a  badger  in  1782.  He  named  the  parasite  Ascaris 
criniformis.  Froelich,  in  1789,  found  hookworms  in  the  fox  and  called  them  hook- 
worms from  the  hook-like  ribs  of  the  copulatory  bursa.  He  proposed  the  generic 
name  Uncinaria.  Therefore  Uncinaria  belongs  to  the  hookworms  of  the  fox  and 
is  not  valid  for  any  human  species. 

In  1838,  Dubini  found  a  hookworm  as  a  human  parasite.  On  account  of  the 
four  ventral  teeth  projecting  from  the  mouth  he  gave  it  the  name  Agchylostoma  or 
correctly  Ancylostoma. 

Bilharz  and  Griesinger  noted  the  connection  of  the  parasite  with  Egyptian 
chlorosis,  but  it  was  not  until  the  time  of  the  St.  Gothard  tunnel  (1880),  that  the 
importance  of  the  parasite  was  recognized.  Grassi  noted  the  diagnostic  value  of 
the  ova  in  faeces  in  1878.  In  1902,  Stiles  noted  and  described  the  hookworm  found 
in  the  United  States  as  different  and  proposed  the  name  Uncinaria  americana,  later 
changed  to  Necator  americanus.  A.  J.  Smith  had  also  recognized  the  morphological 
differences. 

Hookworms  may  be  found  in  the  small  intestine  (jejunum)  of  man 
in  enormous  numbers.  They  either  produce  their  effects  by  feeding  on 
the  mucosa  or  by  causing  loss  of  blood. 

The  males  are  little  more  than  1/3  of  an  inch  (9  mm.)  long  and  the  females  little 
more  than  1/2  inch  (13  mm.)  in  length.  The  male  can  readily  be  distinguished 
by  his  umbrella-like  expansion  or  copulatory  bursa.  The  tail  of  the  female  is  pointed. 
The  vulva  of  A.  duodenale  is  located  in  lower  half  of  the  ventral  surface;  that  of 
N.  americanus  in  upper  half.  The  large  oval  mouth  of  the  Old  World  hookworm 
has  four  claw-like  teeth  on  the  ventral  side  of  the  buccal  cavity  and  two  on  the  dorsal 
aspect.  In  N.  americanus  the  buccal  capsule  is  round,  smaller  and  the  ventral 
teeth  are  replaced  by  chitinous  plates.  Dorsally  there  are  two  similar  but  only 
slightly  developed  lips  or  plates.  A  very  prominent  conical  dorsal  median  tooth 
projects  into  the  buccal  cavity.  Through  it  passes  the  duct  of  the  dorsal 
cesophageal  gland.  The  copulatory  bursa  of  the  N.  americanus  is  also  different, 
being  terminally  bipartite  and  deeply  cleft  in  the  division  of  dorsal  ray  rather  than 
tripartite  and  shallow  as  with  the  A.  duodenale. 

The  delicate-shelled  eggs  pass  out  in  the  faeces,  and  in  one  or  two  days  a  rhabditi- 
form  embryo  (200X14/0  is  produced. 


THE   HOOK   WORM 


275 


The  mouth  cavity  of  the  embryo  is  about  as  deep  as  the  diameter 
of  the  embryo  at  the  posterior  end  of  the  mouth  cavity;  that  of  Strongy- 
loides  is  only  about  one-half  as  deep  as  the  diameter. 

A  temperature  of  i°  C.  kills  the  eggs  in  twenty- four  to  forty-eight  hours.  After 
moulting  twice,  it  remains  rather  quiescent  but  still  lying  inside  the  discarded  skin. 
It  reaches  this  stage  in  from  four  to  fourteen  days  according  to  the  temperature. 

The  soil  in  the  area  of  the  hookworm-egg-laden  stool  becomes  infested  with  these 


FIG.  75. — i  a,  Copulatory  bursa  of  Necator  americanus,  showing  the  deep  cleft 
dividing  the  branches  of  the  dorsal  ray  and  the  bipartite  tips  of  the  branches;  also 
showing  the  fusion  of  the  spicules  to  terminate  in  a  single  barb.  Scale  i/io  mm. 
ib,  Branches  of  dorsal  ray  magnified.  2a,  The  buccal  capsule  of  N.  americanus. 
2b,  The  same  magnified.  3a,  Copulatory  bursa  of  Ancylostoma  duodenalc,  showing 
shallow  clefts  between  branches  of  the  dorsal  ray  and  the  tridigitate  terminations. 
Spicules  hair-like.  3b,  The  dorsal  ray  magnified.  4a,  The  buccal  capsule  of  A. 
dnodcnale,  showing  the  much  larger  mouth  opening  and  the  prominent  hook-like 
ventral  teeth.  4b,  the  same  magnified.  $a,  Egg  of  N.  americanus.  5b,  Egg  of 
A.  duodenale.  6a,  Rhabditiform  larva  of  Strongyloides  as  seen  in  fresh  faces. 
6b,  Rhabditiform  larva  of  hookworm  in  faeces  eight  to  twelve  hours  after  passage 
of  stool. 

larvae  which  will  even  climb  up  blades  of  grass.  It  is  for  this  reason  that  children  with 
their  bare  feet  are  so  liable  to  infection.  (If  the  larvae  get  into  water  they  sink  to  the 
bottom.)  It  is  at  this  stage  that  it  burrows  into  the  skin  of  man,  producing  the  so- 
called  "ground-itch  "  at  the  site  of  entrance.  Having  gained  access  to  the  lymphatics 
and  veins,  they  eventually  reach  the  lungs.  Here  they  get  into  the  bronchioles  and 


276  THE   ROUND    WORMS 

undergo  a  third  moulting.  They  then  work  their  way  up  the  trachea  to  the  glottis 
and  are  swallowed  to  then  become  adults  in  the  intestine.  Dr.  Stiles,  while  accept- 
ing this  theory  of  the  life  history,  thinks  it  probable  that  infection  is  also  brought 
about  by  swallowing  directly  some  infecting  stage. 

Very  young  dogs  can  be  infected  with  human  hookworm  larvae,  but  infection  of 
man  with  the  dog  hookworm  (A.  caninum)  has  not  been  reported. 

The  infecting  stage  is  not  a  young  larva  but  one  in  which  the  cuticle 
of  a  former  larval  stage  instead  of  being  cast  off  remains  and  acts  as  a 
protecting  sheath  for  the  more  mature  larva  within.  In  this  stage 
larvae  may  remain  alive  for  six  to  twelve  months  and  have  greater 
powers  of  resistance  than  younger  larvae.  Introduction,  either  by  skin 
or  mouth,  of  these  cuticle-covered  larvae  is  followed  by  finding  of  eggs  in 
the  faeces  in  about  fifty  days. 

It  has  been  claimed  that  where  ordinary  microscopical  examination  for  ova  will 
show  40%  of  infections  and  methods  involving  concentration  55%  that  cultural 
methods  will  show  99%.  A  convenient  method  of  culturing  is  to  make  a  pile  of 
filter-paper  circles  of  2  inches  diameter  and  about  1/4  inch  high  and  place  in  the  center 
of  a  4-inch  Petri  dish.  Fill  the  dish  with  water  about  to  the  height  of  the  filter-paper 
and  spread  a  thick  layer  of  faeces  on  the  top  of  the  filter-paper  island.  The  larvae 
hatch  out  in  about  six  days  and  swim  out  into  the  clear  surrounding  water.  They 
are  best  found  by  centrifuging  the  fluid  containing  them. 

Of  the  three  standard  drug  treatments  that  of  thymol  seems  to  be  preferable 
to  betanaphthol  and  vastly  so  to  eucalyptus  oil.  In  giving  thymol  it  is  imperative 
that  neither  alcohol  in  any  form  nor  fats  in  any  form  be  given  on  the  day  of  treatment. 
Stiles  prefers  to  divide  his  thymol  into  three  doses,  1/3  at  6  A.M.,  1/3  at  7  and  1/3 
at  8  followed  by  epsom  salts  at  10  A.  M.  The  patient  should  be  on  a  restricted 
diet  and  be  given  two  doses  of  salts  on  the  two  days  preceding  the  administration 
of  thymol. 

Necator  americanus. — This  is  the  species  of  hookworm  found  in 
the  southern  states  of  the  United  States  and  the  West  Indies. 

It  is  very  prevalent  in  Guam,  L.  I.  It  was  found  by  Looss  in  pigmies  from 
Central  Africa,  so  that  this  parasite  was  undoubtedly  brought  to  America  by  slaves. 
It  is  not  rare  in  Ceylon,  India  and  the  Philippine  Islands. 

The  copulatory  bursa  of  this  parasite  has  double  spicules  which  fuse  terminally 
in  a  barb,  while  A.  duodenale  has  two  fine  hair-like  spicules.  The  head  of  Necator 
has  a  more  marked  dorsal  bend  than  Ancylostoma. 

To  sum  up  the  differences  between  this  species  and  A.  duodenale 
we  have  with  Necator:  i.  Smaller  oral  cavity  which  shows  rib-like 
projections  leading  to  the  two  ventral  plates  instead  of  the  four  promi- 
nent projecting  teeth.  2.  The  dorso-median  ray  of  the  caudal  bursa  is 
deeply  cleft  and  shows  bipartite  divisions  terminally  instead  of  having 


ASCARID.E 


277 


a  shallow  cleft  and  tripartite  division.     3.  The  vulva  of  the  female  is 
placed  in  the  anterior  third. 

The  eggs  of  N.  americanus  are  larger  than  those  of  A.  duodenale.  In  hook- 
worm disease  we  have  ground  itch,  tibial  ulcer,  anaemia,  interference  with  physical 
and  mental  development  and,  in  bad  cases,  dirt  eating. 

Shade,  moisture  and  sandy  soil  seem  essential  factors  for  the  development  of 
hookworm.  Prophylaxis  is  essentially  one  connected  with  soil  pollution.  The 


Ova  or  the  Parasitic  Worms  or  Man 
NEMATODA 

SCALE.        x      ieoo 

A  A 


ORAWN 


CrVMthout  outer 
envelope  (Modt 

/roin  Stiles. \<t<£.  an 


ArShowind  / 
sides  syifi-f, 
metrically  H 
convex  [{ 

(after  Loos  5.,»«3t 


over  to 
I  show  one 
laid?  flatten 

edloriginoi) 


Oxyuris 

vermiculorisA.B. 


Strongylus 
subtilis 

s,  1995) 


Asccxris"  Atfchvlostoma 

lumbricoides  ^^^^  d^SSSSS/g.., 

A    R   f^  T\  Tr-i/^V-ii  »t-i  t_*       Looss'ws')"  in  fresh  stool 

rrichuris  Strongvloides 

trichium  ste'rcomlis 

(OriginAl»  x^bJ^LnjJiSUS 


w:    Embryo 
M  in  stool 
after  12  to46 
,  --    .   hours. 

Agchylostoma 
duodenale 

.MKHjt, 


IOOS) 


FIG-  76. — ^Nematode  ova. 

subjection  of  the  faeces  to  some  septic  tank  process  is  more  reliable  than  chemical 
disinfection  or  burying  the  faeces  8  to  10  inches  under  ground. 


These  have  three  papillae  around  oral  cavity,  one  dorsal  and  two 
ventral.  The  male  has  two  equal-length  spicules.  An  intermediary 
host  is  not  needed  in  the  life  history  of  this  family. 

Ascaris  Lumbricoides. — The  male  round  or  eel  worm  is  from  5  to 


278  THE    ROUND    WORMS 

8  inches  (18  cm.)  long  and  the  female  from  7  to  15  inches  (30  cm.)  in 
length.     They  are  from  1/7  to  1/4  of  an  inch  (5  mm.)  in  diameter. 

It  is  probably  the  most  common  parasite  of  man,  especially  in 
children  and  as  it  does  not  require  an  intermediate  host  infection  takes 
place  through  food  or  drink  or  by  ringers  of  children  who  have  been 
playing  where  soil  pollution  exists. 

The  normal  habitat  is  the  upper  part  of  the  small  intestine,  hence  the  ease  with 
which  they  are  vomited  up.  The  three  papillae-like  lips  with  a  constriction  just 
behind  are  easily  studied  with  a  hand  glass.  The  very  long,  whitish,  convoluted, 
thread-like  tubes  of  the  uterus  lead  to  the- opening  of  the  vulva  anteriorly  and  ven- 
trally.  The  male  has  two  large  lance-like  spicules. 

The  body  of  the  worm  is  transversely  striated  and  resembles  the  ordinary  earth- 
worm, but  is  more  grayish  than  red.  The  ova  are  very  characteristic  with  a  rough 
mammillated  exterior.  This  at  times  is  shelled  off  and  we  have  a  smooth  egg  which 
may  be  mistaken  for  eggs  of  other  parasites.  The  eggs  leave  the  body  in  the  faeces 


FIG.  77. — Anterior  extremity  of  Ascaris  lumbricoides ;  A,  seen  from  front;  B,   seen 
from  dorsal  surface.     (Tyson  after  Railliet.) 

and  after  a  long  time — a  few  weeks  to  several  months,  according  to  temperature — 
develop  an  embryo  which  remains  in  the  shell  until  swallowed  by  man.  It  is  stated 
that  they  will  remain  alive  for  years.  On  being  swallowed,  the  embryo  leaves  the 
egg  and  we  have  males  and  females  developing  in  the  small  intestine.  In  countries 
where  such  parasites  abound,  as  in  Guam  and  the  Philippines,  the  possibility  of  their 
getting  into  the  peritoneal  cavity  through  operative  measures  on  the  intestine  must 
always  be  thought  of. 

Guiart  considers  it  probable  that  Ascaris  may  suck  blood,  produce  intestinal 
ulcerations  and  bacterial  infections,  and  perforate  intestine.  Their  entrance  into 
bile  ducts  or  into  larynx  (vomited)  must  be  considered. 

At  autopsy  they  may  be  found  perforating  the  appendix  or  even  filling  up  the 
pancreatic  duct. 

Some  think  that  the  symptoms  of  itching  of  nose  and  anus,  vertigo,  or  convul- 
sions and  anaemia  may  be  due  to  a  toxin  secreted  by  the  worm. 

Ascaris  Canis. — This  is  a  parasite  of  the  dog  and  cat,  but  is  occasionally  found 
in  children.  It  is  much  smaller  than  the  A.  lumbricoides — male  is  2  to  3  inches  long, 


THE    PIN   WORM  279 

female  4  to  5  inches  in  length.     The  parasites  are  characterized  by  the  presence  of 
wing-like  projections  from  the  anterior  end  (arrow-like  head). 

Oxyuris  vermicularis. — This  parasite  is  also  known  as  the  pin-worm 
or  seat-worm  and  is  more  frequent  in  children  than  in  adults. 

The  male  is  about  1/6  of  an  inch  long  and  the  female  a  little  less  than  1/2  inch 
in  length.  The  male  has  an  incurved  tail  with  a  single  spicule  and  the  female  a  long 
tapering  tail.  The  vulva  is  in  the  upper  third. 

These  worms  have  a  clear  slightly  bulbous,  pipe  mouth-piece-like  projection 
surrounding  the  three-lipped  anterior  extremity.  There  is  a  well-marked  bulb 
cesophagus. 

The  eggs  are  thin-shelled  plano-convex,  and  show  a  coiled-up  embryo.  After 
ingestion  of  eggs,  the  adults  develop  in  the  small  intestine  where  copulation  takes 
place;  the  males  then  die.  The  fertilized  females  go  to  the  caecum  and  colon  where 
they  remain  until  they  reach  maturity.  At  this  time  the  females  wander  to  the  rec- 
tum where  they  either  expel  their  ova  or  themselves  work  their  way  out  of  the  anus. 
This  usually  occurs  at  night,  and  the  scratching  induced  by  the  itching  causes  the 
eggs  to  be  widely  spread  about  the  region  of  the  anus.  The  worms  may  also  wander 
into  the  vagina,  urethra,  or  under  prepuce.  It  will  be  seen  that  as  a  result  of  the 
scratching,  the  fingers  become  contaminated  with  o^a  which  may  be  carried  to  the 
mouth  and  so  cause  a  fresh  infection,  no  intermediate  host  being  required.  The 
examination  of  the  material  under  the  finger  nails  of  children  harboring  this  parasite 
may  show  eggs  under  the  microscope.  A  knowledge  of  the  life  history — the  early 
location  in  the  small  intestine,  and  later  on  in  the  large — shows  that  treatment  should 
be  dual  in  its  direction — enemata  for  the  gravid  female  in  the  rectum  and  santonin 
and  calomel  for  the  young  adults  in  the  small  intestine. 

The  diagnosis  is  preferably  made  by  examining  the  stools  for  the 
white,  thread-like  females  which  are  expelled  after  a  diagnostic  dose  of 
calomel  and  salts,  rather  than  by  searching  for  the  eggs. 

These  females,  which  are  packed  with  embryo  containing  eggs,  may  be  seen 
wriggling  on  the  surface  of  the  freshly  passed  fasces.  In  handling  these  worms 
one  should  be  careful  as  they  are  apt  to  cause  infection  should  the  eggs  get  on  the 
fingers. 

ACANTHOCEPHALA. 

These  are  called  thorn-headed  worms  on  account  of  a  proboscis  which  projects 
anteriorly  like  a  little  peg. 

There  are  several  rows  of  hooks  surrounding  this  projection  which  are  directed 
backward  to  enable  the  parasite  to  attach  itself  to  the  intestinal  wall.  The  worm 
absorbs  nourishment  through  the  general  body  wall,  there  being  no  alimentary 
canal  or  mouth.  These  worms  are  common  in  hogs.  The  three-shelled  eggs  are  very 
striking  and  the  intermediate  stage  is  in  June  bugs. 

The  Echinorhynchus  or  Gigantorhynchus  gigas. — This  parasite  is  about  6  inches 
(15  cm.)  long  for  the  male  and  10  to  12  inches  (25  cm.)  for  the  female.  It  has 


280  THE    ROUND    WORMS 

transverse  rings  and  resembles  Ascaris  but  is  more  white  in  color.     It  is  said  to  be 
not  uncommon  in  southern    Russia. 

The  Echinorhynchus  or  Gigantorhynchus  moniliformis  might  be  contracted 
by  persons  eating  death-watch  beetles  as  is  sometimes  done  for  the  improvement  of 
the  complexion. 

HIRUDINEA  (LEECHES). 

Hirudo  medicinalis. — This  is  the  leech  used  medicinally  for  the  abstraction  of 
blood.  They  have  a  secretion  which  prevents  coagulation  of  the  blood  so  that  when 
they  are  removed  the  wound  still  continues  to  bleed. 

Limnatis  nilotica. — This  species  has  been  found  in  many  parts  of  Northern 
Africa  and,  gaining  access  to  the  stomach  through  drinking-water,  it  wanders  to 
the  pharynx,  nares,  and  even  trachea.  Manson  refers  to  a  case  of  obstinate  epis- 
taxis  and  headache  caused  by  a  leech  in  the  nostril. 

This  leech  is  about  4  inches  long  (8  to  10  cm.)  and  about  1/2  inch  (1.2  cm.) 
broad.  The  dorsal  surface  is  greenish  brown  with  narrow  orange  brown  borders. 
The  young  leeches  are  only  about  1/8  inch  (  3  mm.)  long  and  taken  in  with  the  drink- 
ing water  may  attach  themselves  to  the  surface  of  some  mucous  membrane  and  after 
some  weeks  reach  adult  size. 

Haemadipsa  ceylonica. — Whese  are  land  leeches  found  in  India,  Philippines, 
Australia,  and  South  America.  They  are  only  about  i  inch  (25  mm.)  long  and  are 
slender.  They  leave  the  damp  earth  to  climb  shrubs  and  from  there  to  drop  on 
animals  or  man  passing  through  the  forest.  The  bites  are  painless,  but  may  be  fol- 
lowed by  ulcers.  They  may  get  into  the  nostrils. 

They  will  even  penetrate  thick  clothing  in  order  to  reach  the  skin. 


CHAPTER  XIX. 
THE  ARACHNOIDS. 

CLASSIFICATION  OF  THE  ARACIINOIDEA. 


Order. 


Acarina 


Family. 

Trombidiidae 
Gamasidae 

Tyroglyphidae 
Sarcoptidas 
DemodicidcTg 
Tarsonemidae 


Subfamily. 


Ixodidae 


Argasinae 


Ixodinae 


Genus. 

Species. 

Trombidium 

T.  holosericeum 

Dermanyssus 

D.  galling 

Tyroglyphus 

/  T.  farinas 
\  T.  longior 

Sarcoptes 

S.  scabiei 

Demodex 

D.  folliculorum 

Pediculoides 

P.  ventricosus 

[Argas 

/  A.  persicus 
\  A.  miniatus 

Ornithodoros 

O.  savignyi 

Ixodes 

I.  ricinus 

Hyalomma 

H.  aegyptium 

Rhipicephalus 

R.  bursa 

Dermacentor 

{D.  reticulatus 
D.  andersoni 

Margaropus 

M.  annulatus 

Amblyomma 

A.  hebrasum 

Hsemaphysalis 

H.  leachi 

SLinguatula 

L.  rhinaria 

Porocephalus 

P.  constrictus 

Linguatulida 


The  class  Arachnoidea  and  the  class  Insecta  belong  to  the  phylum 
Arthropoda.  This  phylum  contains  a  greater  number  of  species  than 
does  any  other  phylum. 

While  the  lobsters,  crabs  and  water  fleas,  which  belong  to  the  class 
Crustacea,  are  important  zoologically,  they  are  of  very  slight  impor- 
tance medically.  Besides  the  Crustacea  we  have  the  thousand-legged 
worms  or  Myriapoda. 

The  different  classes  of  Arthropoda  resemble  the  segmented  worms 
but  have  as  distinction  the  possession  of  jointed  appendages  which 
proceed  from  the  somites  in  pairs.  Some  of  the  pairs  of  limbs  are  for 
locomotion;  at  times,  certain  ones  may  be  specialized  for  food  taking. 

The  somites  or  divisions  of  the  body  have  a  chitinous  exoskeleton. 

281 


282  THE    ARACHNOIDS 

Respiration  takes  place  through  the  medium  of  gills  in  the  Crustacea 
and  by  tracheal  tubes  in  the  Myriapoda,  Arachnoidea,  and  Insecta. 

The  Arachnoidea  have  no  antennae  while  the  Myriapoda  and  Insecta 
have  a  single  pair  of  antennae,  the  former  having  numerous  pairs  of 
legs  or  jointed  appendages  while  the  latter  have  only  three  pairs  of  legs. 
The  Arthropoda  have  segmented  bodies,  but  they  differ  from  the  worms 
in  having  jointed  appendages  for  the  purpose  of  taking  in  food  and 
moving  from  place  to  place.  They  also  have  an  exoskeleton  which  is 
more  or  less  unyielding  from  the  deposit  of  chitin  in  the  cuticle.  This 
cuticle  is  not  a  true  skin  but  only  a  secretion  of  the  epidermis.  . 

Within  this  external  skeleton  we  have  a  dorsal  digestive  system  and 
a  ventral  nervous  system. 

THE  ARACHNOIDEA. 

The  Arachnoidea  differ  from  the  Insecta  in  having  the  head  and 
thorax  fused  together.  They  also  have  four  pairs  of  ambulatory  appen- 
dages, while  the  insects  only  have  three  pairs.  The  Arachnoidea  never 
have  compound  eyes — 'these  when  present  being  simple.  Of  the  two 
orders  of  Arachnoidea  of  interest  medically  the  Acarina  is  far  more 
important  than  the  Linguatulida. 

ACARINA. 

Of  the  acarines  we  are  chiefly  interested  in  the  mites  and  the  ticks. 
The  acarines  do  not  show  any  separation  of  the  abdomen  from  the 
cephalo-thorax.  A  hexapod  larva  develops  from  the  egg;  this  is  suc- 
ceeded by  an  octopod  nymph  which  differs  from  the  adult  in  not  having 
sexual  organs. 

In  addition  to  the  four  pairs  of  legs  in  the  fully  developed  acarine  there  are  two 
other  paired  appendages,  the  chelicerae.  in  front  of  the  mouth,  and  the  pedipalps 
on  either  side  of  the  mouth. 

Trombidiidae. 

These  generally  have  a  soft,  more  or  less  hairy  integument  and  are  often  brightly 
colored.  The  two  eyes  are  often  pedunculated  and  the  cheliceras  are  lancet  shaped 
and  the  palps  project  beyond  the  rostrum  as  claw-like  appendages.  A  tip-like 
appendage  on  the  apical  segment  of  the  palps  is  characteristic.  A  very  common 
and  annoying  member  of  this  family  is  the  hexapod  larva  of  the  Trombidium 
holosericeum.  It  is  usually  designated  Leptus  autumnalis.  Popularly  it  is  termed 
"harvest  mite,"  "red  bug"  or  "jigger."  They  are  found  in  the  fields  in  the  autumn 


ACARINA  283 

and  attack  both  man  and  animals.  The  condition  (itching  and  redness)  produced 
is  at  times  called  autumnal  erythema.  There  is  a  Trombidium  in  Mexico  which 
has  a  predilection  for  the  skin  of  the  eyelids,  prepuce,  and  navel.  The  Kedani  mite, 
an  orange-red  larval  mite  about  250  by  125  microns  is  believed  by  the  Japanese 
authorities  to  bring  about  infection  with  Japanese  river  fever  or  Tsutsugamushi, 
as  the  result  of  transmitting  either  a  bacterium  or  protozoon  by  its  bite.  The 
disease  somewhat  resembles  typhus,  although  an  eschar  at  the  site  of  the  bite  and 
lymphatic  involvement  is  present. 

Gamasidae. 

Of  the  Gamasidae.  which  generally  have  a  hard  leathery  body  and1  styliform 
piercing  chelicerae,  delicate  five  jointed  palps  and  styliform  hypostome,  only  the 
Dermanyssus  gallinse  is  of  interest.  This  coleopterous  mite  infests  chicken-houses 
and  sucks  the  blood  of  the  inmates.  They  will  also  attack  man.  Poultrymen  may 
be  troubled  with  a  sort  of  eczema  on  the  backs  of  the  hands  and  forearms,  similar 
to  scabies,  resulting  from  bites  by  these  mites.  They  measure  350X650^.  They 
have  no  eyes. 

Tyroglyphidae. 

Mites  of  this  family  live  on  cheese,  flour,  dried  fruits,  etc.  They  are  small, 
without  eyes,  and  have  a  smooth  skin  and  a  cone-like  appearance  of  the  mouth 
parts  which  are  largely  formed  by  the  chelate  chelicerae.  They  are  chiefly  of 
importance  because  of  their  being  occasionally  found  in  urine,  faeces,  etc.,  and  being 
striking  objects,  the  question  of  pathogenicity  arises.  The  T.  longior  has  been 
associated  with  intestinal  trouble  (probably  a  coincidence,  patient  having  eaten 
cheese  containing  these  mites). 

Glyciphagi  are  found  in  sugar  and  are  the  cause  of  what  is  known  as  "grocers' 
itch."  Rhizoglyphus  parasiticus  is  reported  to  be  the  cause  of  an  itch-like  affection 
of  the  feet  of  coolies  on  tea  plantations.  To  distinguish:  the  dorsum  of  Glyciphagus 
is  hairy  or  plumose;  Tyroglyphus  has  both  claws  and  suckers  on  tarsi,  while  Rhizo- 
glyphus has  only  claws. 

Sarcoptidae. 

These  are  small  eyeless  mites  with  a  transversely  striated  cuticle.  They  live 
on  the  epidermis  of  man  and  various  animals.  The  rostrum  is  chiefly  made  up  of 
chelate  chelicerae  with  quite  short  three  jointed,  rather  adherent  palpi.  It  is  the  female 
that  makes  the  tunnels  in  the  skin  between  the  fingers,  on  penis,  flexor  surface  of 
forearm,  etc.  The  male  dies  off  after  copulation.  The  female  passes  through  four 
stages:  i.  larva;  2.  nymph;  resembles  adult,  but  has  no  sexual  organs;  3.  the  pubes- 
cent female;  4.  the  egg-bearing  female.  A  pair  of  itch  mites  may  produce  1,500,000 
descendants  in  three  months.  Transference  of  eggs,  larvae  or  pubescent  females 
does  not  seem  to  transmit  scabies.  It  is  the  egg-laden  female  only.  The  human 
itch  mite,  Sarcoptes  scabiei,  is  an  oval  mite,  the  male  is  250X150^;  the  female  is 
about  400X300^.  Besides  the  difference  in  size,  the  male  may  be  distinguished  from 
the  female  by  the  fact  that  the  third  and  fourth  pairs  of  legs  in  the  female  have 


284 


THE    ARACHNOIDS 


bristles,  but  in  the  male,  the  fourth  pair  has  suckers.  The  tunnels  made  by  the 
female  have  the  egg-bearing  female  at  the  blind  end;  scattered  all  along  are  fasces, 
eggs,  larvae;  the  eggs  being  next  the  mother  and  the  more  mature  young  at  the  en- 
trance to  the  gallery.  A  diagnosis  can  be  made  from  the  finding  of  either  eggs  or 
larvae.  The  eggs  are  140^  long  and  hatch  out  in  four  to  five  days.  A  female 
becomes  mature  in  about  two  weeks. 

In  treating  itch  with  sulphur  preparations  the  adult  females  and  immature  itch 
mites  are  killed;  the  eggs,  however,  are  not  affected.  Hence  a  second  treatment 
about  ten  days  after  the  first  is  necessary  to  kill  the  young  mites,  which  have  devel- 


-  l_.  AV  E.R  V  — 


FIG.  78. — Arachnoidea  exclusive  of  ticks.  (ia)  Sarcoptes  scabiei,  female; 
(ib)  S.  scabiei,  male;  (2)  Demodex  folliculorum;  (3)  Trombidium  akamushi,  hexapod 
larva  (Kedani  mite);  (4)  Trombidium  holosericeum  larva  (Leptus);  (5)  Dermanyssus 
gallinae;  (6)  Tyroglyphus  longior;  (ja)  Pudiculoides  ventricosus,  male;  (76)  P.  ventri- 
cosus,  young  female;  (jc)  P.  ventricosus  impregnated  female;  (8)  Porocephalus 
armillatus;  (go)  Linguatula  serrata,  female;  (96)  L.  serrata,  larva. 


oped  subsequent  to  the  first  treatment, 
of  itch  mites. 


Different  animals  have  different  species 


Demodicidae  (Hair  Follicle  Mites). 

Demodex  folliculorum. — This  is  a  vermiform  acarine  about  400^  long;  the  eggs 
are  about  75^  long;  they  chiefly  live  in  the  sebaceous  glands  of  nose  and  forehead. 


Tarsonemidae. 

This  acarine  family  shows  a  complete  sexual  dimorphism.     The  Pediculoides 
ventricosus  is  oval  and  about  125X75,"  for  the  male  which  has  claws  at  the  extremi- 


DCODID^E  285 

ties  of  the  anterior  and  posterior  pairs  of  legs;  the  two  other  pairs  have  hooklets 
and  a  sucking  disc.  The  female  is  about  twice  as  long  but  of  the  same  breadth  as 
the  male,  and  has  claws  only  on  the  anterior  legs. 

The  chelicerae  are  needle  like  with  inconspicuous  palps  and  the  front  and  rear 
pairs  of  legs  are  widely  separated.  The  gravid  female  is  like  a  ball  and  is  about 
IQOO/J.  in  diameter. 

They  live  on  wheat  and  may  be  found  in  wheat  straw,  which,  if  handled,  may  be 
followed  by  a  severe  skin  eruption  with  an  irregular  fever. 

Ixodidae. 

This  family  of  the  Arachnoidea  is  one  of  great  medical  interest  and 
of  growing  importance.  It  has  recently  been  proposed  to  raise  the 
ticks  to  a  superfamily,  Ixodoidea  and  to  divide  it  into  the  families 
Argasidae  and  Ixodidae. 

While  only  proven  the  intermediary  hosts  in  the  case  of  the  organism  of  African 
tick  fever  and  the  as  yet  undiscovered  cause  of  spotted  fever  of  the  Rocky  Moun- 
tains, there  is  considerable  speculation  as  to  the  possibility  of  blackwater  fever  being 
due  to  a  Babesia  (Piroplasma).  Piroplasmata  of  animals  seem  to  be  invariably 
transmitted  by  ticks. 

Very  important  diseases  due  to  these  small  pear-shaped  organisms  within  red 
cells  are  .known  for  various  animals,  the  best  known  being  that  of  cattle  in  Texas 
and  known  as  Texas  fever.  Other  piroplasmata  diseases  are  Rhodesian  fever 
(cattle),  heart  water  (sheep),  and  malignant  jaundice  of  dogs.  In  these  diseases 
there  are  pathological  features  which  resemble  blackwater  fever  of  man. 

It  is  of  interest  to  note  that  it  was  with  the  transmission  of  Texas 
fever  through  an  intermediate  host  (the  tick)  that  Smith  and  Kilborne 
(1889-1893)  established  the  zoological  principle  of  transmission  of 
disease  through  arthropod  intermediary  hosts.  This  led  up  to  the 
work  on  malaria,  yellow  fever,  etc. 

Ticks  differ  from  insects  in  having  four  pairs  of  legs,  only  two  pairs  of  mouth 
parts,  and  no  antennae.  They  differ  from  other  acarines  in  having  a  median  probe- 
shaped  puncturing  organ,  the  hypostome,  which  is  beset  with  numerous  teeth 
projecting  backward,  and  in  possessing  stigmal  plates.  The  head,  or  capitulum, 
or  rostrum,  is  the  part  which  projects  anteriorly  from  the  body.  This  carries  the 
piercing  parts  which  are  the  single  hypostome  or  dart  and  a  pair  of  piercing  chitinous 
structures,  the  chelicerae  which  lie  above  the  hypostome.  As  a  sheath  for  these 
delicate  biting  parts  we  have  a  segmented  pair  of  palpi  or  pedipalps.  The  mouth 
is  a  slit  between  the  chelicerae  and  hypostome. 

Two  depressed  pitted  areas  on  the  dorsal  surface  of  the  capitulum  in  the  adult 
female  are  known  as  porose  areas.  Very  important  structures  are  the  stigmal 
plates.  These  are  striking  mosaic-like  areas  which  are  located  just  posterior  to 
each  hind  leg  in  the  Ixodinae  and  between  the  third  and  fourth  legs  in  the  Argasinae. 


286  THE    ARACHNOIDS 

As  the  greatest  confusion  exists  as  to  the  classification  of  ticks,  Dr.  Charles  W. 
Stiles  has  now  in  hand  a  system  of  classifying  ticks  according  to  the  appearance 
of  these  plates  as  seen  under  the  high  power  of  a  microscope.  There  is  great  varia- 
tion in  the  outline  and  general  picture  of  these  stigmal  plates  in  the  different  species. 
The  stigmal  orifice,  the  opening  of  the  tracheal  system,  is  in  the  center.  The 
Ixodinse  have  a  scutum  or  shield-like  chitinous  structure  on  the  dorsal  surface.  It 
covers  almost  the  entire  back  of  the  tick  in  the  male  and  only  a  small  portion  anteri- 
orly in  the  female.  The  genital  opening  is  toward  the  anterior  part  of  the  ventral 
surface.  The  anus,  with  anterior  or  posterior  anal  grooves,  is  near  the  posterior 
third  of  the  venter.  The  legs  have  six  segments,  the  coxa  being  flattened  out  on  the 
surface  of  the  body  and  the  terminal  tarsus  ending  with  a  pair  of  hooks  and  at  times 
with  a  pulvillus.  The  nymph  has  stigmal  plates  but  has  no  genital  opening  while 
the  larva  has  neither  genital  apertures  nor  stigmal  orifice. 

Life  History  of  Ticks. — -This  varies  greatly  according  to  the  sub- 
family, genus,  and  species.  The  female  Ornithodoros  savignyi  lays 
about  140  eggs.  The  larva  does  not  leave  the  egg,  but  moults  inside, 
and  finally  emerges  as  an  eight-legged  nymph.  It  lives  in  the  dust  in 
the  cracks  of  the  native  huts  and  comes  out  at  night  to  feed  on  the  sleep- 
ing natives.  As  the  possibilities  for  destruction  are  not  so  great  as  with 
many  Ixodinae  the  necessity  for  thousands  of  eggs  is  not  imperative  for 
the  continuation  of  the  species  as  with  the  Ixodinae.  With  some  of 
the  Ixodinae  the  females  lay  from  5000  to  20,000  eggs  during  several 
days  or  weeks  and  then  die.  The  eggs  are  preferably  deposited  near 
grass.  The  egg  stage  lasts  from  two  to  six  months,  when  the  six-legged 
larva  ("  seed  tick  ")  emerges.  It  crawls  up  a  blade  of  grass  and  gets  on  a 
passing  animal.  After  feeding,  or  at  times  without  taking  nourishment, 
the  larva  drops  to  the  ground,  and  changes  to  the  pupal  stage  which  has 
four  pairs  of  legs.  The  pupa  crawls  up  a  blade  of  grass  and  gets  on  a 
passing  animal  (the  second  host).  Feeding,  it  falls  to  the  ground  where 
it  remains  eight  to  ten  weeks.  It  moults  and  develops  into  an  adult 
tick.  These  males  and  females  gain  access  to  a  third  animal  host — • 
the  males  fecundate  the  females,  after  which  the  female  gorges  herself 
with  blood;  afterward  dropping  off  the  animal  and  laying  eggs.  With 
some  ticks  fewer  hosts  suffice. 

Cleland  has  noted  reports  of  serious  symptoms,  chiefly  cardiac  and  visual,  from 
the  bite  of  ticks  in  Australia  (Ixodes  holocyclus).  This  is  exceptional,  however,  as 
the  symptoms  following  the  bites  of  such  ticks  are  only  those  of  skin  irritation. 

Classification  of  Ixodidae. 

Subfamily  Argasinae. — Head  concealed  by  body  when  viewed 
dorsally.  No  scutum.  Stigmal  plates  between  third  and  fourth  legs. 


287 


Adults  have    no  suckers  beneath  claws.     Slight  sexual  dimorphism. 
Anus  near  middle  of  venter.     Skin  rough. 

Genus  Argas. — Body  narrow  in  front.  Margins  thin  and  acute.  No  eyes. 
The  A.  persicus  (Miana  bug)  of  Persia  has  been  supposed  to  be  concerned  in  the 
transmission  of  a  serious  disease.  Rostrum  some  distance  from  anterior  margin. 
It  is  also  called  the  fowl  tick  and  transmits  fowl  spirillosis. 

Genus  OrnUhodoros. — Margins  of  body  rounded.  Skin  has  many  irregular 
tubercles.  Rostrum  even  with  anterior  margin  so  that  ends  of  palpi  slightly  project. 
It  is  the  intermediate  host  of  Spirochaeta  duttoni.  (South  African  tick  fever.) 

O.  moubata  is  very  common  in  Africa  living  in  cracks  in  mud  floors  and  bites 
severely  the  sleeping  natives.  The  larva  makes  its  first  moult  inside  the  egg  so 
that  it  shows  4  pairs  of  legs  when  it  emerges.  Christy  thinks  it  may  transmit 
Filaria  perstans. 

O.  savignyi  has  two  pairs  of  eyes  near  base  of  mouth  parts. 


<f .  X^A^ 


EIG.   79. — OrnUhodoros  moubata.     (Murray  from  Doflein.) 


Subfamily  Ixodinse. — 'Mouth  parts  project  in  front  of  body  when 
viewed  dorsally.  Scutum  present.  Stigmal  plates  posterior  to  fourth 
pair  of  legs.  Adults  have  suckers  beneath  claws.  Skin  finely  striated. 

Anus  behind  middle  of  venter. 

Sexual  dimorphism  marked.  Male  has  well-developed  scutum; 
female  has  porose  areas. 

Section  Ixodce. — Transverse  recurved  preanal  groove  in  female.  Male  has  ventral 
surface  covered  with  chitinous  plates.  No  eyes.  Genus  Ixodes. 

Ixodes  has  long  rostrum  with  slender  palpi — palpi  narrow  at  base,  leaving  gap 
between  them  and  hypostome. 

Section  Rhipicephalus. — No  preanal,  but  postanal  groove  in  female.     Ventral 


288 


THE   ARACHNOIDS 


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LINGUATULIDA  289 

surface  of  male  without  adanal  plates  in  Dermacentor,  Haemaphysalis,  Aponomma 
and  Amblyomma,  but  with  one  or  two  pairs  in  Hyalomma,  Rhipicephalus  and 
Margaropus. 

In  the  genera  Hyalomma,  Aponomma  and  Amblyomma  the  palpi  are  long  and 
slender  and  of  about  uniform  width  of  segments. 

In  Hyalomma  the  segments  of  palpi  are  of  about  equal  length.  In  Aponomma 
and  Amblyomma  the  second  palpal  segment  is  much  longer  than  the  others.  Ambly- 
omma differs  from  Aponomma  in  being  very  ornate  and  in  having  eyes. 

In  the  genera  Haemaphysalis,  Dermacentor,  Rhipicephalus,  and  Margaropus 
the  palpi  are  short. 

Haemaphysalis  has  very  broad  rostrum,  triangular  palpi,  and  no  eyes.  Derma- 
centor has  a  square  rostrum  with  short  thick  palpi,  the  second  and  third  joints  being 
as  broad  as  long.  Dermacentor  andersoni  transmits  spotted  fever  of  the  Rocky 
Mountains — not  D.  reticulatus. 

Rhipicephalus  has  palpi  without  transverse  ridges  and  comma-shaped  stigmal 
plates.  The  stigmal  plates  of  Margaropus  are  nearly  circular  and  the  palpi  have 
acute  transverse  ridges  externally.  Margaropus  annulatus  transmits  Texas  fever 
of  cattle.  This  tick  is  also  called  Boophilus  bovis  or  B.  annulatus.  Some  authors 
term  it  Rhipicephalus  annulatus.  Larvae  developing  from  eggs  of  female  ticks 
which  have  fed  on  cattle  infected  with  Texas  fever  transmit  the  disease  which  is  due 
to  a  protozoon  Babesia  bigemina. 

LINGUATULIDA  (TONGUE  WORMS). 

These  are  vermiform  acarines  more  or  less  distinctly  annulated.  They  have 
retractile  hooks  at  either  side  of  the  elliptical  mouth. 

If  the  hooks  are  to  be  considered  not  as  degenerated  legs  but  antennae  and  palpi, 
then  there  is  no  vestige  of  legs  in  the  adult.  The  sexes  are  separate. 

Linguatula  rhinaria. — This  has  been  observed  in  man  both  in  larval  and  adult 
stages. 

The  male  is  white  and  about  3/4  inch  long  while  the  female  is  about  4  inches 
long,  tadpole  shape,  yellowish  in  color,  and  has  about  ninety  segments,  lives  in  "the 
nasal  cavity  and  frontal  sinus  of  dogs,  rarely  in  horses  and  sheep,  and  very  rarely 
in  man. 

The  female  lays  embryo-containing  eggs  which,  gaining  freedom  through  the 
nasal  mucus,  are  swallowed  by  various  animals.  A  larva  develops  which  bores  its 
way  through  the  gut  and  encysts  in  the  liver  or  mesenteric  glands.  After  several 
moultings,  they  work  their  way  again  to  the  intestines  and  so  get  out  of  the  body 
of  their  host;  or  they  may  wander  to  lungs  and  trachea  and  either  escape  or  take  up 
their  position  in  the  nostrils  to  become  adults  and  produce  eggs.  Consequently, 
one  animal  may  act  as  intermediate  and  definitive  host  or  these  cycles  may  take 
place  in  distinct  animal  hosts. 

The  larval  form  (1/5  in.)  is  far  more  common  in  man  than  the  adult.  Symptoms 
are  referred  to  liver  in  both  larval  and  adult  stage,  and  epistaxis  and  nasal  symptoms 
for  adult  stage  only. 

Porocephalus  constrictus. — The  adult  form  P.  moniliformis  lives  in  the  lungs  of 
snakes  and  the  eggs  are  probably  ingested  by  drinking  water.  These  eggs  develop 


2QO  THE   ARACHNOIDS 

into  a  curled-up,  ringed  larva,  about  1/2  inch  long  with  twenty-three  rings,  which 
is  encysted  especially  in  the  liver  or  lungs.  These  escape  and  are  swallowed  by  the 
snakes,  their  definitive  hosts. 

While  in  the  liver  or  lungs  of  man  the  patient  may  have  signs  of  bronchitis, 
hepatitis  or  peritonitis.  Cases  usually  only  discovered  at  postmortem.  Parasites, 
however,  might  possibly  be  found  in  sputum  or  faeces. 


CHAPTER  XX. 
THE  INSECTS. 

CLASSIFICATION  or  THE  CLASS  INSECTA. 


Order.               Family.              Subfamily.             Genus. 

Species. 

Siphuncu- 

{Pediculus 

P.  capitis 

lata 

Pediculidae 

Phthirius 

P.  vestimenti 

P.  pubis 

Rhynchota 

Acanthiidae 

Acanthia 

A.  lectularia 

(Hemip- 

Conorhinus       \ 

C.  megistus 

tera)           {  Reduviidae 

C.  sanguisuga 

f 

{  Pulex 

P.  irritans 

Xenopsylla 

X.  cheopis 

Siphonap- 

Pulicin 

ae          Ceratophyllus 

C.  fasciatus 

tera 

Pulicidae 

Ctenocephalus 

C.  serraticeps 

[  Ctenopsylla 

C.  musculi 

Sarcops 

>yl-       Sarcopsylla 

S.  penetrans 

linae 

Simulidae 

Simulium 

S.  reptans 

(buffalo  gnats) 

Psychodidag 

Phlebotomus 

P.  papatasii 

(moth  midges) 

Chironomidae 

Ceratopogon 

C.  pulicaris 

(midges)             f  Culicinae          Culex 

C.  fatigans 

Culicidae               \   Anophe-          Anopheles 

A.  maculipennis 

[       linae 

(Tabanus 

T.  bovinus 

Tabanidas 

Haematopota 

H.  pluvialis 

(horseflies) 

1    Pangonia 

P.  beckeri 

(  Chrysops 

C.  dispar 

Diptera 

f 
Glossina 

G.  palpalis 
G.  morsitans 

Stomoxys 

S.  calcitrans 

Muscidae 

Musca 

M.  domestica 

Auchmeromyia 

A.  luteola 

Calliphora 

C.  vomitoria 

Lucilia 

L.  caesar 

Chrysomia 

C.  macellaria 

(screw-worm) 

Sarcophagidae 

iSarcophaga 
Ochromyia 

S.  carnaria 
O.  anthropophaga 

GEstridae 

Dermatobia 
Hypoderma 

D.  cyaniventris 
H.  diana 

2QI 


2Q2  THE    INSECTS 

INSECTA. 

The  class  Insecta  has  one  pair  of  antennas,  three  pairs  of  mouth  parts 
(the  fused  labium  being  considered  as  one  pair),  and  three  pairs  of  legs. 
They  have  three  divisions  of  the  body — head,  thorax,  and  abdomen. 

The  head  carries  the  antennas  and  mouth  parts;  the  thorax,  which  is  divided 
into  the  pro-meso  and  meta  thorax,  carries  upon  the  ventral  surface  of  each  thoracic 
segment  a  pair  of  legs  and  on  the  dorsal  surfaces  of  the  two  posterior  segments  a 
pair  of  wings.  The  abdomen  does  not  support  appendages.  The  air  is  supplied 
by  means  of  tracheae — branching  breathing  tubes  which  have  external  openings  or 
stigmata.  The  tracheae  are  stiffened  by  spiral  chitinous  bands.  The  Malpighian 
tubules  are  excretory  organs  of  the  alimentary  system  and  excrete  nitrogenous 
waste  material.  Insects  have  two  pairs  of  wings,  the  second  pair  of  which  is  fre- 
quently rudimentary  and  shows  simply  as  knob-like  projections.  These  are  termed 
halteres  or  balancers.  In  some  insects  both  pairs  of  wings  are  rudimentary,  as  in 
Siphonaptera. 

Where  insects  show  metamorphosis  we  have  voracious  worm-like  larvas  coming 
out  of  eggs;  these  larvae  are  succeeded  by  a  quiescent  nonfeeding  encased  pupa 
which  finally  develops  into  an  imago  or  fully  developed  insect.  An  insect  which 
does  not  present  this  developmental  cycle  shows  incomplete  matamorphosis.  Of 
the  class  Insecta  only  the  Siphunculata  Rhynchota,  Siphonaptera,  and  Diptera 
are  of  special  importance. 

SIPHUNCULATA. 
These  are  small  flat  wingless  insects  not  showing  metamorphosis. 

The  Pediculidse. 

In  this  family  there  are  no  wings  and  there  is  no  metamorphosis. 
The  acorn-shaped  eggs  (nits)  are  deposited  on  hairs  of  the  host. 

Pediculus  capitis. — The  female  is  about  1/12  of  an  inch  long;  the  male  smaller- 
They  vary  in  color  according  to  the  color  of  the  hair  of  the  host.  The  eggs  are  de- 
posited on  the  hairs  of  the  head  in  number  of  6c  which  hatch  out  in  about  six  days. 
The  thorax  is  as  broad  as  the  abdomen.  The  male  louse  is  rounded  off  posteriorly 
and  shows  a  dorsal  aperture  for  a  pointed  penis,  while  the  female  is  recognized  by  a 
deep  notch  at  the  apex  of  the  last  abdominal  segment.  There  seems  to  be  a  marked 
preference  exhibited  by  lice  for  their  own  peculiar  racial  host.  It  has  recently  been 
suggested  that  this  might  account  for  certain  peculiarities  in  infection  where  different 
races  were  living  together  and  under  similar  conditions  as  to  food  and  environment, 
and  yet  only  one  race  contracts  the  disease  (beriberi).  The  head  louse  has  been 
found  to  harbor  leprosy  bacilli  when  living  on  a  leper. 

Pediculus  vestimenti. — This  louse  lives  about  the  neck  and  trunk  and  deposits 
its  eggs  in  the  clothing.  They  number  about  75  and  hatch  out  in  three  or  four  days 
and  become  mature  in  about  two  weeks.  Unlike  the  fleas  there  is  no  grub  stage. 


LICE 


293 


It  is  almost  twice  the  size  of  the  P.  capitis  and  the  abdominal  seg- 
ment is  broader  than  the  thorax.  The  abdomen  is  less  markedly 
festooned  than  that  of  P.  capitis;  is  less  hairy  and  contains  8  segments 
as  against  6  for  P.  capitis. 

It  has  recently  been  shown  to  transmit  typhus  fever  and  more  recently  Nicolle 
has  demonstrated  it  as  a  carrier  of  relapsing  fever,  the  spirochastes  being  introduced 
by  the  material  from  the  crushed  louse  being  rubbed  into  the  wound  by  the  scratch- 
ing of  the  victim  (just  as  with  the  flea  in  plague)  and  not  by  the  bite  itself. 


FIG.  81. — Siphunculata  and  Rhynchota.  i.  Pediculus  capitis.  2,  Pediculus 
vestimenti.  2a.  Protruded  rostrum  of  Pediculus.  3.  Phthirius  pubis.  4.  Acan- 
thia  lectularia.  5.  A.  rotundata.  6.  Conorhinus  megistus. 

Phthirius  pubis. — This  louse  is  popularly  known  as  the  crab  louse.  The  female 
is  little  more  than  1/25  of  an  inch  in  length,  and  the  male  a  trifle  less.  They  are 
almost  square.  The  second  and  third  pair  of  legs  are  supplied  with  formidable 
hooks.  They  have  a  preference  for  the  white  race  and  live  about  the  pubic  region. 
The  female  lays  about  a  dozen  eggs,  which  hatch  out  in  about  a  week. 


RHYNCHOTA. 

The  Rhynchota  are  insects  possessing  a  sucking  beak  in  which  the 
lower  lip  forms  a  long  thin  tube  or  rostrum  which  can  be  bent  under  the 


294  THE    INSECTS 

head  or  thorax.     Inside  this  tube  are  biting  parts — mandibles  and  max- 
illae.    The  metamorphosis  in  this  order  is  not  marked. 

They  have  no  palpi.  The  lower  lip  or  labium  or  beak  has  it's 
edges  curved  to  form  the  tube  and  it  is  only  covered  by  the  labrum  at 
it's  base.  With  the  Diptera  the  labrum  goes  into  the  formation  of  the 
sucking  tube.  The  mandibles  and  maxillae  are  bristle-like  structures 
serrated  at  the  tip.  The  mandibles  are  grooved  internally  and  form 
when  apposed  a  tube  for  blood. 

The  Acanthiidae. 

These  have  a  flattened  body,  a  three-jointed  rostrum,  and  four- 
jointed  antennae.  Their  wings  are  atrophied. 

Acanthia  lectularia  (Cimex  lectularius). — This  is  the  cosmopolitan  bedbug. 
It  measures  about  1/5  by  1/8  of  an  inch  (5  by  3  mm.).  It  is  of  a  brownish-red  color. 
The  most  conspicuous  feature  of  the  bedbug  is  the  long  proboscis  continuous  with 
the  dorsal  integument  of  the  head  and  tucked  under  the  ventral  surface.  There 
are  two  prominent  eyes  and  two  four-jointed  antennae.  There  are  eight  abdominal 
segments.  The  bedbug  lives  in  cracks  and  crevices,  especially  about  beds.  It  is 
said  thay  can  migrate  from  house  to  house.  At  any  rate,  they  are  frequently  trans- 
ferred with  wash  clothes.  They  have  a  penetrating  odor  when  crushed.  The 
female  deposits  about  fifty  eggs  at  a  time  in  cracks  and  in  ten  days  they  hatch  out 
into  larvae  which  pass  insensibly  into  adults  by  a  series  of  five  moultings;  this  deposit- 
ing of  eggs  occurs  about  four  times  a  year. 

The  bedbug  is  very  probably  the  intermediate  host  in  kala  azar  and  it  has  been 
incriminated  in  connection  with  typhus  fever  and  relapsing  fever. 

In  India  the  A.  rotundata  is  the  one  encountered.  It  is  of  a  dark  mahogany 
color,  has  a  smaller  head,  narrower  abdomen,  thick  rounded  prothoracic  borders 
and  is  more  densely  covered  with  hairs  than  A.  lectularia.  The  prothorax  of  A. 
lectularia  is  flattened  at  the  side. 

* 

Reduviidae. 

These  bugs  have  a  long  narrow  head  and  a  distinct  neck.  The 
antennae  are  long  and  slender.  The  antennae  in  the  genus  Conorhinus 
are  inserted  about  midway  between  the  eyes  and  point  of  the  head. 

Conorhinus  sanguisuga. — This  is  known  as  the  Texas  or  Mexican  bedbug,- 
and  was  formerly  the  foe  of  the  common  bedbug,  but  having  gotten  a  taste  for 
human  blood  through  the  Cimex  or  Acanthia,  it  now  prefers  man.  It  is  extending 
toward  the  North.  It  has  wings.  The  bites  are  much  more  severe  than  those 
of  the  common  bedbug.  It  is  of  a  dark  brown  color,  nearly  an  inch  in  length,  with 
a  long,  flat,  narrow  head  and  a  short  thick  rostrum.  They  can  run  as  well  as  fly. 
They  bite  at  night. 


FLEAS  2Q5 

CONORHINUS  MEGISTUS. — This  is  called  "Barbeiro"  in  Brazil  on  account  of  its 
preference  for  biting  the  face.  The  Schizotrypanum  cruzi  undergoes  a  develop- 
mental cycle  in  this  bug  which  transmits  the  disease. 

SlPHONAPTERA. 

These  are  laterally  flattened  wingless  insects  and  undergo  a  complete 
metamorphosis. 

Pulicidae. 

This  family  is  divided  into  two  subfamilies — the  Pulicinae  and  the 
Sarcopsyllinae.  In  the  former  the  female  remains  practically  unchanged 
after  fecundation,  in  the  latter  the  abdomen  becomes  enormously  dis- 
tended with  eggs,  and  the  female  remains  stationary  after  her  impreg- 
nation in  the  burrow  which  she  has  made  under  the  skin. 

Pulicinae. — Formerly,  with  the  exception  of  infection  with  Dipylidium  caninum, 
the  fleas  were  only  under  suspicion  as  carriers  of  disease;  ideas  having  been  enter- 
tained as  to  their  being  possible  transmitters  of  relapsing  fever,  typhus  fever  and 
kala  azar.  Trypanosoma  lewisi  is  transmitted  by  fleas,  either  Pulex  irritans  or 
C.  canis.  The  trypanosome  undergoes  development  in  the  flea  and  the  infecting 
material  is  in  the  faeces  of  the  flea  and  transmission  occurs  by  the  licking  on  the  part 
of  the  rat  of  faeces  from  an  infected  flea.  The  infection  has  no  connection  with  the 
puncture  wound  of  the  flea  as  is  the  case  with  plague.  As  a  result  of  the  convincing 
experiments  of  the  British  Plague  Commission,  their  role  in  the  transmission  of 
plague  has  been  absolutely  established.  It  is  by  the  bite  of  the  Xenopsylla  cheopis 
that  plague  is  chiefly  transmitted  from  rat  to  rat,  and  in  bubonic  and  septicaemic 
plague  it  is  apparently  the  intermediary  .in  human  infection. 

The  average  capacity  of  a  flea's  stomach  is  about  0.5  cmm.  so  that  with  a  rat 
dying  with  septicaemic  plague  and  with  possibly  100  million  bacilli  to  one  c.c.  of 
blood  the  flea  would  take  in  about  5000  bacilli.  Furthermore  these  multiply  in 
the  alimentary  canal  so  that  the  digested  blood  teems  with  bacilli  when  reaching 
the  anus  of  the  flea.  The  plague  bacilli  are  passed  out  with  the  faeces  and  these 
being  rubbed  into  the  puncture  of  the  flea  bite  bring  about  infection.  The  puncturing 
apparatus  of  the  flea  consists  of  a  pointed  epipharynx  and  two  distally  serrated 
mandibles.  These  chitinous  biting  parts  are  contained  in  the  labium  which  divides 
distally  into  two  labial  palps.  The  maxillae  are  conspicuous  triangular  structures 
and,  projecting  farthest  anteriorly,  are  the  conspicuous  four-jointed  maxillary  palps, 
often  mistaken  for  antennae.  By  the  apposition  of  the  internally  grooved  mandibles 
to  the  epipharynx  a  tube  is  formed  through  which  the  blood  is  sucked  up.  The 
antennae  are  inconspicuous  and  are  in  close  apposition  to  the  sides  of  the  head, 
behind  the  eyes,  and  can  only  be  well  made  out  with  a  lens.  Fleas  have  three  pairs 
of  legs,  and  the  male  can  be  distinguished  from  the  female  by  its  smaller  size  and  the 
conspicuous  coiled-up  penis  within  the  abdomen.  The  female  has  a  conspicuous 
gourd-like  spermatheca  which  varies  in  shape  in  different  species.  The  body  of 


296  THE    INSECTS 

the  flea  is  flattened  laterally.  They  may  or  may  not  have  eyes,  and  certain  con- 
spicuous structures  called  combs  are  of  importance  in  classification.  In  the  meta- 
morphosis of  the  flea  the  eggs  are  hatched  out  in  dust  of  crevices,  etc.,  into  bristled 
larvae  in  about  one  week.  The  larva  forms  a  cocoon  and  develops  into  a  nymph 
which  has  three  pairs  of  legs.  The  nymphs  emerge  from  the  cocoon  as  adult  fleas 
in  about  three  weeks  after  the  larva  forms  it. 

KEY  TO  THE  FLEAS. 

A.  With  combs. 

1.  Eyes  present. 

a.  Combs  along  inferior  border  of  head  and  on  prothorax. 
Ctenocephalus  serraticeps. 

b.  Combs  only  on  prothorax.     Ceratophyllus  fasciatus. 

2.  Eyes  absent. 

a.  Collar  of  combs   on  prothorax  and  four  short  ones   along 
inferior  border  of  head.     Ctenopsylla  musculi. 

B.  Without  combs. 

a.  Ocular  bristle  arises  near  upper  anterior  margin   of   eye.     A 
line  between  this  and  the  oral  bristle  approximately  vertical. 
Two    bristles    posterior    to    antennae.     Xenopsylla     cheopis. 
Lxmopsylla  cheopis.     Formerly  Pulex  cheopis. 

b.  Ocular  bristle  arises  near  lower  anterior  margin  of  eye.     A  line 
between  this  and  the  oral  bristle    approximately   horizontal. 
One  bristle  posterior  to  antennae.     Pulex  irritans. 

The  common  human  flea  of  Europe  is  the  Pulex  irritans;  that  of  the  United  States 
the  Ctenocephalus  serraticeps  or  dog  flea.  The  flea  that  is  implicated  with  plague 
is  the  Xenopsylla  cheopis.  It  resembles  P.  irritans,  but  is  more  yellow  than  brown 
in  color.  It  also  has  a  greater  number  of  bristles  on  the  head.  The  ocular  bristle 
runs  above  and  in  front  of  the  eye;  that  of  P.  irritans  below.  It  is  principally  the 
flea  of  Mus  decumanus,  the  sewer  rat;  but  the  house  rat,  M.  rattus,  becomes  infected 
from  coming  in  contact  with  the  sewer  rat  in  the  basement. 

Ceratophyllus  fasciatus  is  the  common  rat  flea  of  Europe  and  the  U.  S.  In  the 
tropics  X.  cheopis  is  the  common  rat  flea  (98%  in  India).  Ctenocephalus  serrati- 
ceps, Ctenopsylla  musculi  and  Pulex  irritans  have  also  been  frequently  found  on 
both  Mus  norvegicus  and  M.  rattus.  To  distinguish  M.  norvegicus  from  M.  rattus 
we  have  in  the  former  (i)  ears  which  barely  reach  the  eyes  when  laid  forward  and 
(2)  tail  rather  shorter  that  length  of  head  and  body  together  (only  89%  of  length 
of  head  and  body  together).  With  M.  rattus  the  tail  is  longer  than  the  head  and 
body  together  (25%  longer)  and  the  extended  ear  covers  or  reaches  beyond  the 
middle  of  the  eye.  M.  rattus  has  a  sharper  nose,  longer  and  more  delicate  tail 
and  thinner  ears  than  M.  norvegicus  (formerly  M.  decumanus). 

M.  alexandrinus  is  a  variety  of  M.  rattus.     Rats  and  mice  belong  to  the  family 


SARCOPSYLLA 

Muridae  and  the  common  mouse  is  M.  musculus. 
Rodentia  of  the  class  Mammalia. 


297 
They  belong  to  the  order  of 


Sarcopsyllinae. 

Belonging  to  the  subfamily  Sarcopsyllinae,  the  Sarcopsylla  penetrans  (Derma- 
tophilus  penetrans)  is  of  great  importance  in  tropical  countries.  It  is  known  as 
the  chigoe,  nigua,  or  jigger.  The  male  and  virgin  female  are  unimportant 
as  they  do  not  penetrate^  the  skin  but  act  as  ordinary  fleas.  The  female,  which 
when  unimpregnated  is  only  about  1/24  of  an  inch  long,  when'  impregnated 
bores  its  way  into  the  skin  of  man,  especially  about  the  toes,"  soles  of  the 


FIG.  82. — Fleas,  bedbugs  and  ticks.  A,  Lcemopsylla  cheopis;  B,  P.  irritans; 
C,  Ctenopsylla  musculi;  D,  bedbug;  E,  cross  section  of  rostrum  of  Ornithodorus; 
F.  longitudinal  section  of  Ornithodorus. 

feet  or  finger-nails,  and  in  the  chosen  site  develops  enormously,  becoming 
as  large  as  a  small  pea.  This  enlargement  takes  place  in  the  second  and 
third  abdominal  segments  and  is  packed  with  eggs  measuring  about  400  microns 
long  and  numbering  about  100.  A  small  black  spot  in  the  center  of  a  tense  rather 
pale  area  is  characteristic.  The  metamorphosis  is  similar  to  that  of  the  flea.  Sar- 
copsylla can  be  differentiated  from  the  flea  by  the  proportionately  larger  head  to 
the  body,  and  especially  by  the  fact  that  the  head  is  the  shape  of  the  head  of  a  fish, 
distinctly  pointed.  With  the  fleas  the  lower  border  of  the  head  comes  out  in  a 
straight  line  to  join  the  curve  of  the  upper  part.  In  the  Sarcopsylla  lower  and  upper 
border  of  head  are  both  curved. 


298 


THE    INSECTS 


DlPTERA. 

The  insects  of  the  order  Diptera  are  of  great  importance  medically 
in  a  variety  of  ways,  either  by  the  direct  irritation  of  their  bites,  by  their 
transmitting  disease  directly,  as  does  the  common  house  fly  typhoid 
fever,  or  by  acting  as  intermediate  hosts  for  various  parasites.  They 
are  characterized  by  mouth  parts  formed  for  puncturing,  sucking,  or 
licking.  They  present  a  complete  metamorphosis,  larva,  pupa,  and 
imago.  As  a  rule,  the  Diptera  have  a  distinct  pair  of  wings,  the  second 
pair  being  rudimentary.  With  the  Aphaniptera  or  Siphonaptera  the 


FIG.  83. — i  and  2,  male  and  female  Xenopsylla  cheopis.     3,  Head  of  Cerato- 
phyllus.     4  and  5,  male  and  egg  distended  female  of  Sarcopsylla  penetrans. 

wings  are  practically  absent.     Under  the  Aphaniptera,  we  have  to 
consider  the  Pulicidae  or  flea  family. 

The  order  Diptera  is  usually  divided  into  the  following  suborders:  i.  Orthorrha- 
pha:  Diptera  with  larvae  having  a  differentiated  head.  The  imago  breaks  through 
the  larval  or  pupal  case  by  a  T-shaped  break  and  has  no  frontal  lunule  (an  oval  space 
just  above  the  root  of  the  antennae).  The  Orthorrhapha  are  divided  into:  a.  Nemo- 
cera  (with  long,  many  jointed  antennae)  and  b.  Brachycera  (with  short  antennae) 
2.  Cydorrhapha:  larvae  without  differentiated  head.  The  imago  escapes  through 
an  anterior  opening  and  has  a  lunule  and  ptilinum  (an  inflatable  projecting  organ 


BITING   FLIES  2  99 

just  above  the  root  of  the  antennae).  If  the  halteres  are  covered  by  a  scale  (squama) 
we  have  calyptrate  Cyclorrhapha;  if  not,  acalyptrate.  These  squamae  are  large 
enough  in  the  calyptrate  species  to  even  conceal  the  halteres  when  the  fly  is  looked 
at  from  above.  3.  Pupipara:  the  larvae  are  extruded  ready  to  begin  the  pupal  state. 

The  males  of  flies  where  the  two  compound  eyes  come  together  above  the  antennae 
are  referred  to  as  holoptic,  if  more  or  less  widely  separated  as  dichoptic.  Ocelli  are 
three  single  eyes  usually,  when  present,  situated  in  the  triangular  space  between  the 
compound  eyes  in  the  front  (the  space  separating  the  compound  eyes). 

In  studying  the  biting  flies  it  is  very  important  to  recognize  the  anterior,  small, 
or  mid-cross  vein.  This  short  transverse  rib  or  vein  is  the  key  to  wing  venation. 
Beneath  it  is  the  discal  cell  and  it  bounds  the  first  posterior  cell  internally  or  basally. 
It  is  also  of  great  value  in  differentiating  Culicidae.  The  character  of  the  antennae 
should  also  be  noted  carefully.  The  study  of  the  bristles  about  head,  thorax,  and 
abdomen  (chaetotaxy)  is  more  difficult.  Anyone  taking  up  the  study  of  flies  should 
carefully  note  the  wings,  etc.,  of  Musca  domestica.  By  putting  a  few  house  flies 
on  moist  horse  manure  in  a  gauze-covered  bottle  the  entire  metamorphosis  may  be 
observed. 

Tabanidae. 

This  is  the  family  of  horseflies,  gadflies,  breeze  flies  or  green-headed  flies.  It 
is  the  most  numerous  family  of  the  Diptera — there  being  more  than  1000  species. 
The  females  are  blood  suckers;  the  males  live  on  flowers  and  plant  juices.  The  eyes 
are  usually  very  brilliant  in  color,  and  in  the  male  make  up  the  greater  part  of  the 
head. 

They  belong  to  the  suborder  Orthorrhapha  and  in  the  group  of  short  antennae 
flies  (Brachycera).  Five  posterior  cells  are  always  present. 

The  antennae  consist  of  three  segments.  No  arista.  The  epipharynx  is  tube 
like,  the  hypopharynx  has  a  groove  and  both  are  awl  shaped.  The  pair  of  maxillae 
are  serrated  and  the  mandibles  lancet  like.  They  have  rather  coarse  maxillary 
palps.  The  labellae  are  prominent  at  the  extremity  of  the  fleshy  labium.  They  are 
thick  set  flies  and  rarely  show  color.  The  body  of  the  larva  has  eleven  segments 
with  a  small  but  distinct  head.  The  eggs  are  deposited  in  masses  on  the  leaves  or 
stems  of  plants  about  marshy  places.  The  larva  is  carnivorous. 

Tabanus  autiunnalis. — Is  about  3/4  of  an  inch  long;  it  is  dark  in  color,  and  has 
four  longitudinal  bands  on  the  thorax.  The  last  joint  of  the  antennae  has  a  crescentic 
notch.  The  wings  do  not  overlap. 

Hsematopota  pluvialis. — In  the  Haematopota  there  is  no  crescentic  antennal 
notch,  and  the  wings  overlap.  The  abdomen  is  narrower  than  in  Tabanus.  The 
brimp,  one  of  the  Haematopota,  bites  man  severely. 

Pangonia  beckeri. — The  genus  Pangonia  is  characterized  by  a  very  long,  slender, 
and  more  or  less  horizontal  proboscis. 

Chrysops  dispar. — Chrysops  has  three  ocelli,  in  this  respect  differing  from  the 
genera  Tabanus  and  Haematopota.  The  wings  are  widely  separated  and  spotted. 
The  antennae  of  Chrysops  are  especially  long  and  slender.  Chrysops  and  Haemato- 
pota produce  the  greatest  amount  of  pain  from  their  bites.  The  Tabanidae  are  not 
implicated  as  intermediate  hosts  in  the  transmission  of  disease.  By  their  bites, 


300 


THE    INSECTS 


however,  they  may  transmit  disease  directly,  as  with   anthrax.     Two  species  of 
Chrysops  have  been  found  to  transmit  Filaria  loa. 

Muscidae. 

The  Muscidae,  Sarcophagidae,  and  CEstridae  are  calyptrate  Cyclorrhapha. 

The  common  housefly,  M.  domestica,  is  the  best  example  of  this  family. 

The  arista  is  feathered  both  dorsally  and  ventrally  with  straight  hairs.  The 
fourth  longitudinal  vein  bends  down  in  a  rather  sharp  angle  as  compared  with 
Stomoxys,  which  gives  the  first  posterior  cell  rather  a  fusiform  appearance.  The  eyes 
are  close  together  in  the  male,  far  apart  in  the  female.  The  female  lays  about  125 


FIG.  84. — Wing  venation  of  A,  Tabanus;  B,  Stomoxys;  C,  Glossina. 

eggs  in  a  heap  preferably  in  fermenting  horse  manure.  The  larva  comes  out  in  about 
thirty-six  hours.  Very  characteristic  are  the  stigmata  decorating  the  blunt  posterior 
ends.  (See  illustration.) 

The  larval  stage  lasts  seven  to  ten  days  and  then  the  barrel- shaped  pupal  stage 
is  entered  upon.  This  lasts  about  three  days  when  the  adult  fly  emerges.  This 
fly  is  incapable  of  biting,  the  piercing  organs  being  fused  with  the  labium,  but  may 
transmit  disease  directly,  carrying  infectious  material  from  the  source,  as  in  faeces, 
to  the  food  about  to  be  ingested.  Their  r61e  in  typhoid  fever  is  one  of  immense 
importance.  By  reason  of  its  hairy  sticky  legs,  habits  of  frequent  defecation  and 
constant  regurgitation  the  housefly  is  an  important  agent  in  the  spread  of  cholera, 
dysentery,  infantile  diarrhoeas  and  tropical  ophthalmias  as  well  as  typhoid. 


BITING   FLIES 


3OI 


In  the  Muscidae  the  antennae  hang  down  in  front  of  the  head  in  three  segments 
and  have  an  arista  plumose  to  the  tip.  The  first  posterior  cell  is  narrowed.  There 
are  no  bristles  on  abdomen  except  at  tip. 

(I)  Stomoxys,    Haematobia   and    Glossina    have   a   more   or   less 
elongated  proboscis  adapted  for  biting.     Stomoxys  has  delicate  palpi, 
shorter  than  the  proboscis,  and  arista  feathered  only  on  the  dorsal  side 
with  straight  hairs.     Haematobia  has  club-like  palpi  about  as  long  as 
proboscis  and  arista  with  hairs  dorsally  and   ventrally.     Glossina  has 
thick  set  but  not  clubbed  palpi  and  an  arista  feathered  on  the  dorsal 
side  with  branching  hairs. 

(II)  Musca,  Calliphora,  Chrysomyia,  Lucilia,  and  Cordylobia  do 
not  have  a  proboscis  adapted  for  biting. 


FIG.  85. — Common  housefly  (Musca  domestica):  Puparium  at  left;  adult  next, 
larva  and  enlarged  parts  at  right.  All  enlarged.  From  circular  71  (by  L.  O. 
Howard),  Bureau  of  Entomology,  U.  S.  Department  of  Agriculture. 

Stomoxys  calcitrans. — These  greatly  resemble  the  common  housefly  in  size 
and  shape.  They  can  be  easily  distinguished  by  the  black,  piercing  proboscis  extend- 
ing beyond  the  head.  There  are  longitudinal  stripes  on  the  thorax  and  spots  on  the 
abdomen.  The  proboscis  on  examination  will  be  seen  to  be  bent  at  an  angle  near  its 
base.  The  palps  are  short  and  slender.  The  wings  diverge  widely. 

The  female  lays  about  60  banana-shaped  eggs  in  horse  manure.  These  hatch 
out  in  three  days  as  larvae  which  turn  into  pupae  in  two  or  three  weeks.  After  about 
ten  days  the  fly  emerges.  The  genus  Stomoxys  includes  vicious  biters.  This  is 
the  fly  which  comes  into  houses  before  a  rain,  and  which  has  given  the  common  house- 
fly the  reputation  of  biting  before  a  rain.  Stomoxys  may  be  implicated  in  trans- 
mitting surra  (Trypanosoma  evansi). 

It  has  now  assumed  great  importance  as  a  transmitter  of  poliomye- 
litis and  possibly  of  pellagra. 


302 


THE   INSECTS 


The  horsefly  (Haematobia  irritans)  rarely  bites  man.  In  these  the  palpi  are  much 
longer  than  in  Stomoxys,  being  as  long  as  proboscis.  These  palps  are  also  thick 
and  spatulate. 

Glossina  palpalis. — This  is  the  tsetse  fly  that  is  responsible  for  the 
transmission  of  human  trypanosomiasis  (sleeping  sickness). 

The  tsetse  fly  is  a  small  brownish  fly  about  1/3  of  an  inch  long.  The  pro- 
boscis extends  vertically  and  has  a  bulb  at  its  base.  The  arista  is  plumose 
only  on  the  upper  side  and  the  individual  hairs  are  themselves  feathered. 
The  wings  are  carried  flat,  closed  over  one  another  like  the  blades  of  a  pair  of  scissors 


FIG.  86. — Insects  in  which  the  adult  stage  is  important,  (i)  Stomoxys  cal- 
citrans;  (2)  S.  calcitrans,  larva;  (3)  Tabanus  bovinus;  (4)  Tabanus  larva;  (5)  Glossina 
palpalis;  (6)  G.  palpalis,  side  view;  (7)  G.  palpalis  pupa;  (8)  Glossina  palps  and  arista. 

and  project  beyond  the  abdomen.  The  most  characteristic  feature  of  the  tsetse 
fly  is  the  way  the  fourth  longitudinal  vein  bends  up  abruptly  to  meet  the  mid  cross 
vein  and  then  curves  downward  to  run  parallel  with  the  third  longitudinal  vein. 
In  Stomoxys,  the  wings  separate;  in  Haematopota  they  just  meet,  and  in  Glossina 
they  cross.  Glossinae  bite  chiefly  in  the  daytime. 

The  tsetse  fly  does  not  lay  eggs,  but  gives  birth  to  a  single  full-grown  larva 
almost  as  large  as  the  mother  which  immediately  bores  its  way  into  the  soil  and 
becomes  a  pupa. 

The  pupal  stage  is  about  a  month  and  the  larval  stage  in  the  mother  about  two 
weeks.  G.  palpalis  bites  in  the  day  time.  Both  males  and  females  bite.  Glossina 


BLOW   FLIES 


303 


morsitans  transmits  the  cattle  trypanosome  disease,  nagana  and  the  human  infection 
due  to  Trypanosoma  rhodesiense. 

Auchmeromyia  luteola. — This  is  an  African  fly,  the  larva  of  which  is  known  as 
the  "  Congo  floor  maggot,' '  and  is  a  blood  sucker.  The  larva  is  of  a  dirty- white  color 
and  about  2/3  of  an  inch  long.  It  crawls  out  at  night  and  feeds  on  the  sleeping 
natives.  This  is  the  only  known  instance  of  a  blood-sucking  larva. 

Calliphora  vomitoria  and  Lucilia  caesar. — These  are  flies  with  brilliant  metallic- 
colored  abdomens,  commonly  called  blow  flies  in  the  case  of  Calliphora  and  blue- 
bottle flies  for  Lucilia.  They  deposit  their  eggs  on  tainted  meat  and  in  wounds. 


11 


FIG.  87. — Insects  in  which  the  larval  stage  is  important,  (i)  Chrysomyia 
macellaria;  (2)  C.  larva;  (3)  Dermatobia  cyaniventris  larva,  early  stage  (ver  ma- 
caque); (4)  D.  cyaniventris  larva,  later  stage  (torcel  or  berne);  (5)  D.  cyaniventris; 
(6)  Auchmeromyia  luteola;  (7)  A.  luteola,  larva;  (8)  Sarcophaga  magnifica;  (9) 
S.  magnifica  larva;  (10)  Anthomyia  pluvialis;  (n)  A.  pluvialis  larva. 

« 

Many  cases  of  obscure  abdominal  trouble  are  probably  due  to  the  larvae  of  these 
flies.  Intestinal  myiasis  is  undoubtedly  of  greater  importance  than  has  been 
thought.  The  larvae,  with  hook-like  projections  anteriorly  and  a  ringed  body,  can 
easily  be  recognized  in  the  faeces.  They  have  been  mistaken  for  flukes.  They 
also  have  a  tendency  to  be  attracted  by  those  with  ozena  and  the  larvae  may  develop 
in  the  nostrils. 

Chrysomyia  macellaria. — This  is  known  as  the  screw-worm  when  in  the  larval 
stage.  The  adult  fly  resembles  the  blue-bottle  flies.  It  is  distinguished  from  them, 
however,  by  the  presence  of  black  stripes  on  thorax.  These  flies  are  very  common 
over  nearly  all  North  and  South  America.  The  thorax  is  striped.  The  eggs,  which 


304  THE    INSECTS 

number  250  or  more,  when  deposited  in  the  nostrils  or  in  wounds,  develop  into  the 
screw-worm  larva,  which  may,  by  going  up  into  the  frontal  sinus,  cause  death. 
These  larvae  have  twelve  segments  with  rings  of  minute  spines. 

Ochromyia  anthropophaga  (Cordylobia  anthropophaga  or  Tumbu  Fly). — This 
is  an  African  fly  whose  larvae  develop  under  the  skin  of  man  and  animals.  It  is 
known  as  the  Ver  de  Cayor.  The  larva  resembles  the  Ver  Macaque,  is  rather 
barrel  shaped  and  beset  with  small  spines.  It  bores  its  way  into  the  skin  and  makes 
a  lesion  like  a  boil  which  has  a  central  opening  through  which  the  larva  breathes. 

Sarcophagidae. 

These  are  known  as  "flesh  flies."  The  most  important  characteristic  is  the  fact 
that  the  arista  is  plumose  up  to  the  mid-point,  beyond  which  it  is  bare.  They  are 
usually  thick  set  and  moderately  large  flies. 

Sarcophaga  carnaria. — This  is  a  grayish  fly  with  three  stripes  on  thorax  and 
black  spots  on  each  segment  of  the  abdomen.  It  is  viviparous.  The  larvae  gain 
access  to  nasal  and  other  cavities  and  there  develop.  Cases  of  death  have  been 
reported.  Naturally,  the  fly  deposits  its  larvae  on  decaying  flesh. .  In  times  of  war 
all  of  these  flies  become  important  by  reason  of  "maggots"  in  the  wound.  These 
larvae  are  the  most  common  ones  in  intestinal  myiases.  The  mouth  booklets  are 
strongly  curved  and  separate.  Each  abdominal  segment  has  a  girdle  of  spines. 
The  anterior  end  is  somewhat  pointed.  The  hind  stigmal  plate  is  in  a  deep  cavity. 

(Estridae. 

The  flies  of  this  family  are  usually  called  botflies.  The  mouth  parts  are  almost 
vestigial.  They  have  a  large  head  with  a  somewhat  bloated-looking  lower  portion. 
They  are  often  rather  hairy.  The  larvae  which  develop  from  the  eggs  are  parasitic 
either  in  the  alimentary  canal  or  the  subcutaneous  tissues. 

Dermatobia  cyaniventris. — These  are  large,  thick-set  flies  about  3/5  inch  long, 
with  prominent  head  and  eyes,  small  antennae,  and  a  marked  narrowing  at  the 
junction  of  thorax  and  abdomen.  The  thorax -is  grayish  and  the  abdomen  a  metallic 
blue.  The  larvae  are  deposited  under  the  skin  in  various  parts  of  the  body.  When 
the  larvae  move  they  cause  considerable  pain.  At  first  the  larva  is  club-shaped, 
but  later  on  it  becomes  oval.  The  former  is  called  Ver  Macaque,  the  latter  Torcel. 

Hypoderma  diana. — The  larval  form  of  this  fly  has  been  reported  three  times 
for  man.  It  forms  tumors  under  the  skin  which  it  is  thought  may  reach  this  location 
by  proceeding  in  some  way  from  the  alimentary  canal. 

In  Hypoderma  the  arista  is  bare  while  in  Dermatobia  the  upper  border  is  plumose. 


CHAPTER  XXI. 
THE  MOSQUITOES. 

MOSQUITOES  (Culicidae)  are  of  the  greatest  importance  medically, 
not  only  from  their  influence  upon  health  in  general  by  reason  of  inter- 
ference with  sleep  and  possibly  from  direct  transmission  of  disease,  but, 
more  specifically,  they  are  the  only  means  by  which  it  at  present  appears 
possible  to  bring  about  infection  with  such  diseases  as  yellow  fever, 
malaria,  filariasis,  and  possibly  dengue.  In  addition,  many  diseases 
of  animals  are  transmitted  by  mosquitoes. 

The  Culicidae  differ  from  all  other  Diptera  in  having  scales  on  their 
wings  and  generally  on  head,  thorax,  or  abdomen. 

To  identify  a  mosquito,  examine  a  wing  and  note  the  scales;  also  note  the  presence 
of  two  distinct  fork  cells  and,  in  addition,  that  the  costal  vein  passes  completely 
around  the  border  of  the  wing,  making  a  sort  of  fringe  with  its  scales.  Mosquitoes 
undergo  a  complete  metamorphosis,  there  developing  from  the  egg  a  voracious, 
rapidly-growing  larva;  next,  a  nongrowing,  nonfeeding  stage — the  pupa  or  nymph. 
There  the  head  and  thorax  are  combined  in  an  oval  body,  from  the  back  of  which 
projects  the  siphon  tubes;  and  tucked  in  ventrally  is  a  small  tail- like  appendage. 

The  fully  developed  insect  emerges  from  the  pupa. 

The  Culicidae  belong  to  the  suborder  Nematocera.  These  have  long  articulated 
antennae  and  include  four  families:  Culicidae  Chironomidae,  Simulidae,  and  Psycho- 
didae. 

The  principal  mosquito-like,  blood-sucking  Diptera  which  are 
frequently  mistaken  for  mosquitoes — 'none  of  which  have  scales  on  their 
wings — are  the  following: 

1.  Chironomidae  or  Midges. — The  blood-sucking  species  of  Chironomidae,  which 
are  found  in  most  parts  of  the  world,  belong  chiefly  to  the  genus  "  Ceratopogon." 
These  midges  are  of  very  small  size,  about  1/12  of  an  inch  long,  are  able  to 
get  through  netting  and,  usually  being  in  swarms,  they  are  exceedingly  trouble- 
some.    The  antennas  have  thirteen  joints  and  the  wings  are  shorter  than  the 
abdomen  and  have  only  longitudinal  veins.     One  of  the.  midges,  the  "jejen" 
of  Cuba,  is  a  great  scourge,  its  small  size  enabling  it  to  enter  eyes  and  nostrils. 
The  larva  of  Chironomus  is  a  red  worm-like  creature;  the  pupa  has  a  tufted 
head. 

2.  Simulidae  or  Buffalo  Gnats. — These  are  small  blood-thirsty  insects  only  about 
1/8  of  an  inch  in  length.     The  thorax  is  humped,  the  legs  are  short  and  the 

20  305 


3°6 


THE    MOSQUITOES 


proboscis  short  and  inconspicuous.  The  antennas  have  eleven  joints  but 
are  rather  short.  One  species,  the  S.  damnosum,  known  by  the  natives  of 
Uganda  as  "Mbwa,"  is  greatly  dreaded;  its  bites  causing  swellings  and  sores. 
Sambon  has  considered  Simulium  reptans  as  the  transmitting  agent  of  pellagra. 
Psychodidse  or  Moth  Flies. — These  are  small,  hairy,  slender  midges,  with 
long  legs  and  a  short  proboscis.  The  antennae  are  long,  hairy  and  consist 
of  12  to  1 6  joints.  Palpi  4  jointed.  They  are  only  about  1/12  of  an  inch  in 
length.  The  hairy  wings  have  numerous  longitudinal  veins.  Some,  as 


FIG.  88. — Mosquito-like  insects  belonging  to  families  Chironomidas,  Simulidae 
and  Psychodidae.  (i)Phlebotomus  papatasii;  (2)  P.  papatasii  (natural  size);  (3) 
P.  papatasii  (larva);  (4)  P.  papatasii  larva  (natural  size);  (5)  Ceratopogon  pulicaris; 
(6)  C.  pulicaris  (natural  size);  (7)  Chironomus  larva;  (8)  Attitude  of  a  Simulium; 
(9)  Simulium  reptans;  (10)  Larvae  of  Simulium. 

Phlebotomus,  have  an  enlongated  proboscis  and  are  vicious  blood  suckers. 
It  has  been  suggested  that  they  may  be  of  importance  in  the  transmission  of 
tropical  ulcer.  A  fever  of  about  three  days'  duration  found  in  Bosnia,  char- 
acterized by  leukopenia  and  similar  to  dengue  and  known  as  Phlebotomus 
or  Pappataci  fever,  has  been  thought  to  be  caused  by  the  bite  of  infected  P. 
papatasii. 

Phlebotomus  is  common  in  the  tropics  and  may  transmit  surra.  The  pro- 
boscis is  much  shorter  than  that  of  mosquitoes. 

Mosquitoes  have  three  main  parts  of  the  body — 'the  head,  the 
thorax,  and  the  abdomen.  On  the  head,  the  space  behind  the  two 


MOSQUITOES 


307 


compound  eyes  is  called  the  frons,  in  front,  and  the  occiput  posteriorly. 
The  nape  is  back  of  the  occiput.  The  bulbous  prolongation  of  the  frons  which 
projects  over  the  attachment  of  the  proboscis  is  the  clypeus.  The  clypeus  is  hairy 
in  the  Culex;  scaly  in  Stegomyia.  The  proboscis  is  straight  in  all  mosquitoes  of 
importance  medically.  It  consists  of  a  fleshy,  scaled,  gutter-shaped  portion  be- 
neath, known  as  the  labium,  which  terminates  in  two  hinge-joint  processes — the 
labella.  At  the  end  of  the  labium  is  a  thin  membrane  (Button's  membrane).  It 
is  through  this  that  filarial  embryos  are  supposed  to  pass  on  their  way  from  the 
interior  of  the  labium  to  enter  the  person  bitten.  The  labium  may  be  considered 
as  the  sheath  of  a  knife,  holding  and  protecting  the  slender,  blade-like  penetrating 


FIG.  89. — Anatomy  of  mosquito,  i,  Dorsal  view  of  mosquito;  2,  wing  of  mos- 
quito; A,  costal  vein;  B,  mid  cross  vein;  C,  posterior  cross  vein;  D,  first  fork-cell; 
E,  second  fork-cell;  3,  various  types  of  scales;  a,  flat  head  scales;  b  and  c,  Mansonia 
wing  scales;  d,  upright  forked  head  scales;  e,  f,  g  and  h,  various  shapes  of  thoracic 
scales. 

organs.  Lying  in  this  groove  we  have,  from  above  downward,  the  horseshoe- 
shaped  labrum — epipharynx,  the  under  surface  of  which  is  open.  This  when  closed 
by  the  underlying  hypopharynx  forms  a  tube  through  which  the  blood  is  sucked 
up  by  the  mosquito.  In  the  hypopharynx,  which  somewhat  resembles  a  hypoder- 
mic needle,  is  a  channel,  the  veneno-salivary  duct.  It  is  down  this  channel  that 
the  malarial  sporozoite  passes.  There  are  two  pairs  of  mandibles  and  two  pairs 
of  maxillae  on  either  side  of  the  hypopharynx — the  mandibles  above  and  the  maxillae 
below.  The  serrations  of  the  maxillae  are  coarser  than  those  of  the  mandibles. 
The  sensory  organs,  the  palps,  lie  on  either  side  of  and  slightly  above  the  proboscis. 


3o8 


THE    MOSQUITOES 


These  are  of  the  utmost  importance  in  differentiating  mosquitoes  and  must  not 
be  confused  with  the  antennae,  which  are  attached  above  the  palpi  and  at  the  sides 
of  the  clypeus.  These  antennae  are  of  importance  in  distinguishing  the  sex  of  the 
mosquito. 

The  thorax  is  largely  made  up  of  the  mesothorax,  at  the  posterior  margin  of 
which  is  a  small,  sharply-defined  piece,  the  scutellum;  this  may  be  smooth  or  trilo- 
bed.  Underneath  and  posterior  to  the  scutellum  is  the  metanotum;  the  metano- 
tum  is  bare  in  Culicinae,  has  hairs  in  Dendromyinae  and  scales  in  Joblotinae. 

There  is  a  pair  of  wings  attached  to  the  posterior  part  of  the  mesothorax  and, 


FIG.  90. — Distinguishing  characteristics  of  mosquito  larvae  and  fly  antennae. 
Siphon  tubes  of  i,  Stegomyia,  2,  Culex,  3,Tasniorhynchus;  mental  plates  of  4,  Taenio- 
rhynchus,  5,  Stegomyia,  6,  Culex;  larval  antenna- of  7,  Culex,  8,  Stegomyia,  9,  Anoph- 
eles; antennae  of  10,  Muscidae,  n,  Tabanidae,  12,  Simulidae,  13,  Sarcophagidae. 

more  posteriorly  still,  a  pair  of  rudimentary  wings  (halteres)  attached  to  the 
metanotum.  The  three  pairs  of  legs  are  attached  to  the  thorax. 

There  are  nine  segments  in  the  abdomen.  The  genitalia  arise  from  the  termi- 
nal segments  as  bilobed  processes.  In  the  male  there  is  a  pair  of  hook-like  appen- 
dages or  claspers,  between  which,  and  ventrally  situated,  are  the  harpes,  also  a  pair 
of  chitinous  processes. 

In  considering  the  question  of  the  possible  danger  which  might  arise  from  the 
introduction  of  a  case  of  yellow  fever,  malaria,  or  filariasis,  it  would  give  the  greatest 
information  if  mosquito  ova  were  at  hand  so  that  we  could  by  watching  the  develop- 
ment from  egg  to  larva,  pupa,  and  insect,  have  all  the  points  from  which  to  decide 
as  to  the  genera  developing  in  the  given  locality.  It  is  generally  a  very  easy  matter 


MOSQUITO 


309 


to  dip  out  large  numbers  of  larvae  from  the  pools  and  having  noted  the  character- 
istics of  the  larvae,  to  do  the  same  when  the  pupae  develop;  so  that  we  have  only  to 
verify  our  identification  when  the  insect  emerges  from  the  pupa. 

THE  OVA. 

The  egg  raft  of  Culex,  containing  about  250  ova,  is  quite  perceptible  on  the 
surface  of  the  water  as  a  black,  scooped-out  mass,  about  1/5  of  an  inch  in  length. 
The  eggs  are  set  vertically  in  the  raft.  The  eggs  of  the  Stegomyia  are  laid  singly 
and  have  a  pearl-necklace-like  fringe  around  them. 


ATE      H/MRS 


~--  "  UATERAL.  ABDOM.  HAIRS 


ANTENNA 


MOUTH  BRUSH 


FIG.  91. — i.  Asiphonate  larva.     Anopheles.     2.  Siphonate  larva.     Stegomyia. 

The  Anophelinae  eggs  are  oval  in  shape  with  air-cell  projections  from  either  side. 
They  are  laid  in  triangle  and  ribbon  patterns.  The  markings  of  these  air  cells 
vary  and  have  been  used  for  differentiation.  The  length  of  time  of  the  egg  stage 
varies  according  to  temperature  and  other  conditions — one  to  three  days  for  Stego- 
myia and  two  to  four  days  for  Anophelinae.  The  Anophelinae  are  more  difficult 
to  raise  than  Culex  or  Stegomyia. 


LARV.E. 


The 


There  are  two  great  classes  of  larvae — the  siphonate  and  the  asiphonate. 
latter  are  always  Anophelinae. 

The  Culicinae  larvae  have  a  projecting  breathing  tube  at  the  posterior  extremity 
which  is  called  a  respiratory  siphon.     This  projects  off  at  an  angle  from  the  axis 


3io 


THE    MOSQUITOES 


of  the  body,  the  true  end  of  which  terminates  in  four  flap-like  paddles.  If  you  di- 
vide the  length  of  the  siphon  by  the  breadth,  you  get  what  is  known  as  the  siphon 
index.  In  Culex  the  siphon  is  long  and  slender,  in  Stegomyia  it  is  short  and  barrel- 
shaped.  When  at  the  surface  the  Culex  larva  has  its  siphon  almost  vertical  and  the 
body  at  an  angle  of  about  45°. 

The  Stegomyia  larva  hangs  more  vertically.  As  a  rule,  the  hairs  proceeding 
from  the  sides  of  Culex  larvae  are  straight  and  the  head  relatively  large.  There 
are  also  no  palmate  hairs  along  the  sides. 

The  Anophelinae  larvae  have  a  small  head  which  is  capable  of  being  twisted 
around  with  lightning-like  rapidity.  They  are  darker  in  color  and  have  no  siphon; 


^''°" 


Fig.  92. — Metamorphosis  of  mosquitoes,  i,  2,  3,  4,  and  5,  Eggs,  larva,  pupa, 
and  heads  of  male  and  female  Culex;  6,  7,  8,  9,  and  10,  eggs,  larva,  pupa,  and  heads 
of  male  and  female  Anopheles;  n,  12,  13,  14,  and  15,  eggs,  larva,  pupa,  and  heads 
of  male  and  female  Stegomyia. 

float  parallel  to  the  surface  of  the  water;  have  long  lateral  branching  hairs,  and  on 
the  sides  of  each  of  the  five  or  six  middle  abdominal  segments  they  have  a  pair  of 
palmate  hairs.  These  palmate  hairs  are  supposed  to  aid  them  in  keeping  their 
position  on  the  surface  of  the  water.  The  larvae  are  usually  called  "wigglers." 
The  duration  of  the  larval  stage  is  from  i  to  2  weeks,  according  to  the  temperature. 

THE  PUP.E. 

These  have  a  bloated-looking  cephalo-thorax  and  a  shrimp-like  tail — the  latter 
the  abdomen.  Very  important  in  examining  them  with  a  lens  is  to  note  the  char- 
acteristics of  the  siphon  tubes  which  project  from  the  dorsal  surface.  These  siphons 


DISSECTION   OF   THE   MOSQUITO 


311 


are  long  and  slender  in  Culex  and  project  from  the  posterior  portion  of  the  head 
end.  In  Anophelina?  they  are  broadly  funnel-shaped  and  arise  from  the  middle 
of  the  head  end.  The  siphon  of  the  Stegomyia  is  triangular. 

The  bulbous  end  of  the  Culex  nymph  is  more  vertical  than  the  horizontally- 
placed  cephalo-thorax  of  Anopheles.  The  duration  of  pupal  life  is  short — only 
one  to  three  days.  At  the  end  of  this  time  the  pupa  comes  to  the  surface  and 
straightens  out.  The  integument  then  splits  dorsally  and  the  perfect  insect  emerges. 
It  dries  its  wings  for  a  time  on  its  raft-like  pupal  skin  and  then  flies  away. 

From  the  above  it  will  be  seen  that  the  stages  in  the  metamorphosis  of  the  mos- 
quito take  about  two  weeks:  one  to  three  days  for  egg  stage;  seven  to  ten  days  for 
larval  stage,  and  two  to  three  days  for  pupal  stage. 

DISSECTION  or  THE  MOSQUITO. 

The  easiest  way  to  secure  a  mosquito  for  dissection  is  to  use  an  ordinary  plugged 
test-tube.  Slipping  the  open  end  of  the  test-tube  over  the  resting  mosquito, 


FIG.  93. — Anatomy  of  mosquito,  i,  Cross  section  of  proboscis  of  mosquito;  2, 
anatomy  of  mosquito,  longitudinal  section;  3,  tip  of  proboscis  of  mosquito;  a,  labrum- 
epipharynx;  b,  hypopharynx;  c,  manidible;  d,  maxilla. 

by  a  slight  movement,  the  insect  will  fly  toward  the  bottom.  Then  quickly  insert 
the  plug.  If  it  is  not  desired  to  study  the  scales,  the  best  way  to  kill  the  mosquito 
is  by  striking  the  tube  sharply  against  the  thigh.  If  it  is  also  desired  to  study  the 
scale  characteristics  it  is  better  to  put  a  drop  or  so  of  chloroform  on  the  lower 
part  of  the  cotton  plug.  The  vapor  falls  to  the  bottom  of  the  tube  and  kills  the 
mosquito.  Take  the  mosquito  out,  pull  off  legs  and  wings,  and  then  place  the 
body  in  a  drop  of  salt  solution  on  a  slide.  Bile  has  been  recommended.  Then 
hold  the  anterior  end  of  the  thorax  by  pressure  of  a  needle.  With  a  needle 


312  THE    MOSQUITOES 

in  the  other  hand,  gently  crush  the  chitinous  connection  between  the  sixth 
and  seventh  segments  of  the  abdomen.  Then  holding  the  thorax  firm,  steadily 
and  gently  pull  the  last  segments  in  the  opposite  direction.  If  this  is  done 
properly,  a  delicate  gelatinous  white  mass  will  slowly  float  out  in  the  salt 
solution.  One  should  be  able  to  secure  the  alimentary  canal  as  far  up  as 
the  proventriculus,  which  is  just  anterior  to  the  stomach,  the  part  in  which  the 
malarial  zygotes  develop.  Proceeding  from  before  backward,  we  have  the 
proventriculus,  which  is  a  sort  of  muscular  ring  at  the  opening  of  the  stomach 
or  mid-gut.  Leading  from  the  stomach  we  have  the  hind-gut,  which  ends  in  the 
rectum.  Taking  origin  at  the  posterior  end  of  the  stomach  and  festooning  the  hind- 
gut  are  five  longitudinal  tubes — the  Malpighian  tubules.  These  are  characterized 
by  large  granular-like  cells  with  a  prominent  refractile  nucleus.  They  are  re- 
garded as  the  renal  structures.  It  is  in  these  tubules  that  the  embryo  of  the  Filaria 
immitis  of  the  dog  develops.  In  the  female  mosquito,  the  parts  withdrawn  may 
seem  to  be  largely  made  up  of  the  white  oval  ovaries.  These  are  connected  with 
the  spermathecae,  in  which  the  spermatozoa  are  stored  after  fecundation  by  the 
male.  In  the  male  the  testicles  are  quite  distinct.  Next  to  the  examination  of  the 
stomach  for  zygotes,  which  appear  as  wart-like  excrescences  on  its  outer  sur- 
face, the  most  important  structures  are  the  salivary  glands,  where  the  malarial 
sporozoites  are  found.  The  easiest  way  to  dissect  out  the  salivary  glands  is  to 
press  down  firmly,  but  gently,  on  the  anterior  part  of  the  thorax,  and  then  with  the 
shaft  of  a  second  needle,  pressing  on  the  head  to  gently  draw  the  head  away  from 
the  thorax,  so  that  by  this  expression  and  traction  movement  you  extract  them 
with  the  head  segment.  They  are  very  minute  and  are  to  be  told  by  their  ex- 
ceedingly highly  refractile  appearance.  To  stain  for  sporozoites,  pick  up  the  head 
end,  and  with  forceps  draw  the  severed  neck  along  a  clean  dry  slide,  trying  at  the 
same  time  to  smear  out  the  adherent  salivary  glands.  After  drying,  stain 
with  Wright's  stain.  The  sporozoites  are  narrow  falciform  bodies  about  I2/J.  in 
length,  with  a  central  chromatin  dot. 

A  matter  about  which  there  is  dispute  is  as  to  whether  the  salivary  glands 
communicate  with  the  alimentary  canal.  Theobald  states  that  there  is  no  con- 
nection between  them. 


DIFFERENTIATION  OF  CULICIN^E  AND  ANOPHELIN^E. 

It  is  impossible  even  for  an  entomologist  to  differentiate  mosquitoes 
without  recourse  to  elaborate  keys  and  tables.  It  is  a  comparatively 
easy  matter,  however,  to  decide  as  to  whether  the  mosquito  is  a  probable 
malaria  transmitter  or  not. 

While  certain  characteristics  of  the  male  are  used  to  separate  the 
^Edinae  from  other  subfamilies,  yet  it  is  only  with  the  female  that  we 
concern  ourselves  in  differentiating  the  Culicinae  from  the  Anophelinae. 
Therefore,  it  is  first  necessary  to  distinguish  the  male  from  the  female. 
If  the  antennae  have  not  been  torn  off,  this  can  be  decided  by  the  highly 
adorned  plumose  antennae  of  the  male,  those  of  the  female  being  sparsely 


CLASSIFCATION   OF   MOSQUITOES 

decorated  with  short  hairs.  The  palpi  of  the  Anophelinae  tend  to  be 
clubbed,  while  those  of  the  Culex  are  straight.  If  the  antennae  have 
been  broken  off,  look  for  the  claspers  at  the  end  of  the  abdomen. 
Having  determined  that  the  insect  is  a  female,  we  then  proceed  to  place 
it  either  in  the  subfamily  Culicinae  or  Anophelinae  by  a  study  of  the 
relative  length  of  the  palpi  to  the  proboscis.  If  the  palpi  are  shorter 
than  the  proboscis,  it  belongs  to  the  Culicinae;  if  as  long  or  longer,  to 
the  Anophelinae.  The  palpi  of  the  female  Megarhininae  are  also  long, 
but  the  proboscis  is  curved. 

Having  settled  on  the  subfamily,  we  separate  the  genera  by  con- 
sidering such  points  as  character  and  distribution  of  scales  on  back  of 
head,  wings,  thorax,  and  abdomen;  banding  of  proboscis,  legs,  abdomen, 
and  thorax,  shape  of  scales  on  wings,  and  location  of  cross-veins. 


FIG.  94. — Anopheles.  FIG.  95. — Culex. 

Resting  positions  of  anopheles  and  culex  insects.     (Drawn  by  C.  0.  Waterhouse.} 

In  the  resting  position  Culex  allows  the  abdomen  to  droop,  so  that  it  is  parallel 
to  the  wall.  The  angle  formed  by  the  abdomen  with  head  and  proboscis  gives  a 
hunchback  appearance. 

Anopheles  when  resting  on  a  wall  goes  out  in  a  straight  line  at  an  angle  of  about 
45°.  It  resembles  a  bradawl. 

Classification. 

There  are  four  subfamilies  of  Culicidae,  differentiated  according  to 
the  palpi: 

i.  Palpi  as  long  or  longer  than     i.  Palpi  as  long  as  proboscis  in  females;  proboscis 
proboscis  in  male.  straight.     Anophelincs. 

2.  Palpi  as  long  or  shorter  than  proboscis  in  females; 
proboscis  curved.     Megarrhinina. 

3.  Palpi  shorter  than  proboscis  in  females.  Culicina. 

>.  Palpi  shorter  than  proboscis  in  male  and  female. 


THE    MOSQUITOES 


The  important  ones  from  a  medical  standpoint  are  the  Anophelinae 
and  Culicinae. 


Anophelinae. 


i.'  Scales  on  head  only;  hairs 
on  thorax  and  abdomen. 


2.  Scales  on  head  and  thorax 
(narrow  curved  scales). 
Abdomen  with  hairs. 


Scales  on  head  and  thorax 
and  abdomen.  Palpi 
covered  with  thick  scales. 


1.  Scales  on  wings,  large  and  lanceolate.     Anopheles, 
Palpi  only  slightly  scaled. 

2.  Wing  scales  small  and  narrow  and  lanceolate. 
Myzomyia.     Only  a  few  scales  on  palpi. 

3.  Large  inflated  wing  scales.     Cydoleppteron. 


i.  Wing  scales  small  and  lanceolate.     Pyrelophorus. 


i.  Abdominal  scales  only  on  ventral  surface. 
Thoracic  scales  like  hairs.  Myzorhynchus. 
Palpi  rather  heavily  scaled. 


2.  Abdominal  scales  narrow,  curved  or  spindle- 
shaped.  Abdominal  scales  as  tufts  and  dorsal 
patches.  Nyssorhynchus. 


Abdomen  almost  completely  covered  with  scales 
and  also  having  lateral  tufts.     Cellia. 


4.  Abdomen  completely  scaled.     Aldrichia. 


NOTE. — Of  the  above  genera  only  Cydoleppteron  and   Aldrichia  are  unproven 
malarial  transmitters. 

The  Megarhininae  are  of  no  importance  medically. 
The  genus  Megarhinus  has  the  following  characteristics: 

1.  Large    mosquitoes    with    brilliant    metallic    coloring.     (Elephant    mos- 
quitoes.) 

2.  Long,  curved  proboscis. 

3.  Caudal  tufts  of  hairs  on  each  side  of  abdomen. 


The  ^Edinae  are  not  known  to  play  any  role  in  transmission  of  diseases.  This 
subfamily  is  characterized  by  having  the  maxillary  palpi  much  shorter  in  both 
males  and  females  than  the  proboscis. 

One  genus  Sabethes  is  very  characteristic,  owing  to  dense  paddle-like  scale 
structures  on  two  or  more  legs. 


STEGOMYIA 


315 


Differentiation  of  Culicinae  Genera. 


i.  Posterior  cross-vein  nearer 
the  base  of  the  wing  than 
the  midcross-vein. 


1.  Proboscis  curved  in  female.    Psorophora. 

2.  Proboscis  straight  in  female. 

A.  Palps  with  three  segments  in  the  female. 

a.  Third  segment  somewhat  longer  than 
the  first  two.     Culex, 

b.  The   three   segments   equal   in   length. 
Stegomyia. 

B.  Palps  with  four  segments  in  the  female. 

a.  Palps  shorter  than  the  third  of  the  pro- 
boscis.    Spotted  wings.     Theobaldia. 

b.  Palps  longer  than  the  third  of  the  pro- 
boscis.    Irregular  scales  on  w  i  n  g  s  . 
Mansonia, 

C.  Palps   with   five   segments   in   the   female. 
Taniorhynchus. 


2.  Posterior  cross- vein  in  line  with  midcross-vein.     Joblotina. 

3.  Posterior  cross- vein   further  from  base  of  wing  than  midcross-vein. 


Mucidus. 


Of  the  Culicinae  the  genus  Stegomyia  is  of  importance  on  account 
of  yellow  fever.  The  totally  efficient  hosts  for  filariasis  (filarial  embryos 
found  in  the  thorax  and  proboscis)  are  chiefly  among  the  genus  Culex. 
The  genera  Mansonia  and  Taeniorhynchus  may  also  transmit  filariasis. 
Some  think  the  Anophelinae  genera  "Cellia"  and  "Myzomyia"  may 
transmit  filariasis  as  well  as  malaria. 

The  genus  Culex  is  implicated  in  dengue. 

Stegomyia. — This  is  the  most  important  culicine  genus.  These  are  mosquitoes 
with  silver  markings.  The  head,  entirely  covered  with  flat  scales,  has  also  some 
upright  forked  scales.  Scutellum  has  dense  flat  scales.  S.  calopus  is  deep  blackish- 
brown  with  two  thoracic  parallel  lines  with  curved  silver-white  lines  outside  (lyre 
marking).  Banding  of  thorax,  abdomen,  and  legs. 

S.  calopus  bites  only  at  night  after  the  first  feeding.  The  first  meal  of  blood 
however  may  be  taken  in  the  day  time.  To  become  infected  it  must  take  blood 
from  a  yellow-fever  patient  in  the  first  two  or  three  days  of  the  disease.  After 
sucking  the  blood  of  a  yellow-fever  patient  the  mosquitoes  cannot  transmit  the 
disease  by  biting  a  nonimmune  to  yellow  fever  for  a  period  of  eleven  days.  After 
this  time  the  mosquito  remains  infective  for  its  life — in  one  instance  57  days. 

S.  scutellaris  has  a  single  silver  stripe  down  the  center  of  thorax.  Mosquitoes 
of  this  genus  are  often  called  "Tiger  mosquitoes."  The  larvae  have  short,  barrel- 
shaped  siphons.  They  breed  particularly  in  receptacles  about  the  house. 

S.  pseudoscutellaris,  which  resembles  S.  scutellaris,  but  has  white  bands  only, 
at  the  sides  of  the  abdominal  segments,  is  thought  to  transmit  filariasis  in  Fiji. 

Cidex. — Male  palpi  long  and  acuminate.     Head  has  narrow  curved  and  up- 


316  THE    MOSQUITOES 

right  forked  scales.  Laterally,  flat  scales.  C.  fatigans  supposed  to  carry  dengue 
as  well  as  Filaria  bancrofti.  It  also  transmits  Proteosoma  of  birds,  the  life  history 
of  which  in  this  mosquito  paved  the  way  to  the  epochal  discoveries  in  connection 
with  malarial  transmission  by  anophelines.  This  is  a  brown  mosquito  with  pale 
yellow  banding  of  each  abdominal  segment.  The  legs  are  brown  except  for  the 
coxae  and  femora. 

Theobaldia. — These  Culicinas  have  spotted  wings  resembling  Anophelmas. 
These  spots  are  due  to  aggregations  of  scales,  not  to  dark  scales.  Male  palps  are 
clubbed  (like  Anopheles). 

Mucidus. — This  genus  has  a  mouldy  look  from  long  twisted  gray  scales.  The 
legs  are  densely  scaled. 

Mansonia. — This  genus  is  characterized  by  broad  flat  asymmetrical  wing  scales. 
As  the  wing  scales  are  brown  and  yellow  the  wings  are  mottled. 

Grabhamia. — Wings  have  pepper-and-salt  appearance  with  short  fork  cells. 

Taniorhynchus. — This  genus  is  characterized  by  dense  wing  scales,  which  are 
broadly  elongated  with  truncated  apex. 

Acartomyia. — Much  like  Grabhamia,  but  scales  of  head  give  ragged  appearance. 
Male  palpi  clubbed. 

A.  zammittii  was  supposed  to  be  concerned  in  Malta  fever,  but  it  is  now  known 
that  transmission  is  by  medium  of  milk  of  infected  goats. 


CHAPTER  XXII. 
POISONOUS  SNAKES. 

SNAKES  belong  to  the  class  Reptilia  and  the  order  Ophidia.  They 
are  divided  into  colubrine  snakes  (Colubridae)  and  viperine  snakes 
(Viperidae). 

Of  the  Colubridae  the  Hydrophinas  or  sea-snakes  with  rudder-like  compressed 
tail  and  the  Elapinae  with  round  tails  are  most  important. 

Many  of  our  harmless  snakes  such  as  the  garter-snake  and  blacksnake  belong 
to  the  Colubridae. 

The  cobras  belong  to  the  subfamily  Elapinae  and  are  best  known  by  a  neck-like 
expansion  or  hood.  The  only  poisonous  colubrine  snakes  in  the  United  States  are 
the  beadsnake  (Elaps  fulvius)  often  called  the  Florida  coral  snake,  and  the  sonoran 
coral  (Elaps  euryxanthus). 

The  beadsnake  is  black  with  about  seventeen  broad  crimson  bands,  which 
bands  are  bordered  with  yellow. 

Although  small,  they  are  very  venomous.  The  upper  jaw  has  anteriorly  grooved 
fangs,  which  appendages  are  not  present  in  the  nonpoisonous  coral  snakes,  these 
latter  having  teeth  in  the  upper  jaw  so  that  the  wound  shows  four  rows  of  punc- 
tures instead  of  two  rows  and  one  larger  puncture  on  each  side  to  mark  the  entrance 
of  the  fangs. 

In  Asia  there  are  many  important  poisonous  colubrine  snakes;  the  cobra  (Naja 
tripudians),  the  King  cobra  (Naja  bungarus)  and  the  Kraits  (Bungarus  fasciatus). 

All  of  the  Australian  poisonous  snakes  are  colubrines. 

The  Viperidae  which  are  characterized  by  a  triangular  head  and  tubular  poison 
fangs  are  the  most  important  poisonous  snakes  in  America.  The  rattlesnake 
(Crotalus),  the  copperhead  (Agkistrodon),  and  the  water  moccasin  being  widely 
distributed  in  the  United  States. 

There  are  many  harmless  snakes  which  more  or  less  resemble  these  "Pit  Vipers" 
as  the  rattlers,  moccasins,  and  copperheads  are  called.  This  term  refers  to  a  deep 
hole  or  pit  found  on  the  side  of  the  head  between  the  nostril  and  the  eye.  It  is  a 
blind  sac. 

Some  divide  the  Viperidae  into  the  Crotalinse,  which  possess  the  pit  and  the 
Viperinte  which  do  not  have  this  structure. 

The  poison  fangs  are  grooved  or  perforated  and  connected  with  the  poison 
glands  which  resemble  salivary  glands  and  may  be  almost  an  inch  in  length  in  large 
snakes.  The  tongue  is  slender  and  forked  and  is  a  tactile  organ. 

The  jaws  are  remarkable  for  their  great  extensibility,  not  only  vertically,  but 
laterally,  by  the  ligamentous  connections  of  the  two  halves  of  the  mandible  or 
lower  jaw. 

317 


318  POISONOUS    SNAKES 

As  the  fangs  are  directed  backward  it  is  necessary  for  the  snake  when  striking 
to  open  widely  the  jaws  and  bend  back  the  neck.  The  fangs  are  then  brought 
forward  and  erected  by  the  spheno-pterygoid  muscles.  The  snake  bite  is  a  com- 
bination of  bite  and  blow.  The  functional  fangs  of  colubrine  snakes  however  are 
not  mobile. 

In  addition  to  the  possession  of  the  pit,  these  vipers  have  a  more  or  less  trian- 
gular head  and  in  particular  a  single  row  of  large  scales  on  the  under  surface  posterior 
to  the  vent  (anus) ,  while  the  harmless  snakes  show  an  elongated  oval  head  and  two 
rows  of  large  ventral  scales  posterior  to  the  vent. 


FIG.  96. — i,  Single  row  of  scales  posterior  to  vent  (poisonous  snakes — water 
moccasin);  2,  double  row  scales  of  harmless  snake  (Natrix);  3  and  5,  side  and  dorsal 
view  of  head  of  pit  viper;  4  and  6,  side  and  dorsal  view  of  head  of  harmless  snake 
(Natrix) ;  7  and  9,  bite  puncture  and  skull  of  Elaps;  8  and  10,  bite  puncture  and  skull 
of  harmless  snake. 


In  examining  the  wound  made  by  a  snake  the  two  punctures  of  the 
fangs  indicate  the  bite  of  a  poisonous  snake.  If  these  fang  puncture 
points  are  far  apart  it  shows  that  a  large  snake,  and  probably  one 
capable  of  injecting  a  greater  amount  of  venom  has  given  the  bite. 

When  a  snake  strikes  the  fangs  move  from  the  horizontal  to  the  erect  position, 
the  mouth  being  widely  open.  When  the  fangs  enter  the  jaws  close  and  pressure 
is  exerted  on  the  poison  glands  so  that  the  venom  pours  out. 

The  amount  of  venom  varies  with  the  size  and  condition  of  the  snake,  an  adult 
cobra  yielding  about  i  c.c. 


SNAKE  VENOM  319 

The  cobra,  after  having  bitten,  remains  attached  for  a  short  time  while  the 
daboia  strikes  with  the  greatest  rapidity  and  immediately  releases  itself. 

Cobra  and  krait  bites  (colubrine  snakes)  produce  more  or  less 
similar  symptoms  such  as  paralysis  of  articulation  with  nausea  and 
vomiting  and  later  paralysis  of  the  respiratory  apparatus.  There  is 
only  an  insignificant  reaction  at  the  point  of  bite. 

The  venom  is  mainly  neurotoxic,  causing  death  by  paralysis  of  cardiac  and  re- 
spiratory centers.  Cobra  venom  is  also  very  haemolytic.  This  haemolysin  is  acti- 
vated by  the  normal  complement  of  the  serum  of  the  animal  poisoned,  the  haemolysin 
as  contained  in  the  venom  not  being  toxic  when  alone.  Lecithin  also  has  the 
property  of  activating  the  hemolytic  amboceptor  of  venom. 

In  rattlesnake  bites  (viperine  snakes)  there  is  marked  pain  at  the 
site  of  the  wound  with  much  swelling  and  haemorrhagic  infiltration. 
The  swelling  and  petechial  mottling  spread  up  the  limb  from  the  point 
of  entrance  of  the  venom.  Cold  sweats,  nausea,  weak  heart,  and  syn- 
cope are  common. 

Rattlesnake  venom  is  active  chiefly  on  account  of  it's  haemorahagin  or  rather 
endotheliolysin,  which  destroys  the  endothelial  lining  of  blood-vessels. 

Venoms  may  also  contain  proteolytic  ferments  which  may  account  for  the 
softening  of  muscles  in  snake  bite  cases.  The  toxic  effect  of  the  venom  takes  place 
without  an  appreciable  incubation  period,  hence  different  from  true  toxins. 

The  most  venomous  snakes  seem  to  be  the  sea-snakes  (Enhydrina).  This 
venom  is  almost  entirely  neurotoxic. 

The  tiger  snake  of  Australia  is  almost  equally  venomous  and  the  krait  (B. 
cceruleus)  next.  The  rattlesnake  is  about  one-fifth  as  venomous  as  the  krait. 

Certain  venoms  greatly  increase  the  coagulability  of  the  blood  so  that  intravas- 
cular  thromboses  may  occur.  It  is  chiefly  with  the  venoms  of  Daboia  and  Bun- 
garus  that  such  thromboses  are  likely  to  occur  and  this  accounts  for  the  almost 
instantaneous  death  which  at  times  results  from  bites  of  such  snakes. 

The  nonspecific  treatment  of  snake-bite  poisoning  is  i.  by  applying  a  tight 
ligature  above  the  site  of  the  bite.  The  ligature,  which  should  preferably  be  a 
rubber  band,  is  to  be  applied  about  a  single  bone  extremity,  not  about  one  with 
two  supporting  bones.  2.  The  making  of  deep  incisions  about  the  fang  punctures 
and  thorough  irrigation  with  a  strong  solution  of  potassium  permanganate.  Rogers 
has  recommended  that  the  punctures  be  enlarged  with  a  lancet  and  the  resulting 
wound  packed  with  crystals  of  permanganate. 

Recently  Bannerman  has  shown  that  a  dog  bitten  by  a  cobra  cannot  be  saved 
by  free  incision  and  the  rubbing  in  of  permanganate  crystals.  It  may  however  be 
saved  by  the  immediate  injection  of  10  c.c.  of  a  5%  solution  of  permanganate,  but 
not  if  two  minutes  has  elapsed.  Bites  from  the  daboia  are  fatal,  however  the  per-' 
manganate  be  applied. 

He  therefore  does  not  consider  the  permanganate  treatment  of  any  practical 
value.  Rogers  thinks  that  Bannerman's  experiments  with  dogs  do  not  give  a  true 


320  POISONOUS    SNAKES 

idea  of  the  value  of  permanganate  because  he  has  had  success  in  experimenting  with 
cats  and  because  it  has  saved  human  lives.  Chromic  acid  injections  (i%)  have 
also  been  recommended. 

Internally  alcohol  does  not  seem  to  be  of  any  value,  in  fact  many  of  the  deaths 
have  been  attributed  to  excessive  ingestion  of  whiskey.  Strychnine  in  large,  almost 
poisonous  doses,  was  highly  recommended  in  Australia  but  the  statistics  seem  to 
make  the  value  of  this  remedy  doubtful. 

Antivenins. — The  active  agents  of  snake  venoms  may  be  either  of  the  nature 
of  haemorrhagins,  neurotoxins,  or  fibrin  ferments.  In  colubrine  snakes  the  neuro- 
toxin  vastly  predominates  while  with  the  viperines  it  is  the  haemorrhagin.  Certain 
Australian  snakes  contain  all  three  bodies  in  about  equal  proportion  while  with 
the  rattlesnakes  of  America  it  is  almost  entirely  the  haemorrhagin  which  causes 
the  poisoning.  The  Elaps  of  Florida  is  a  colubrine  snake  and  its  venom  is  neuro- 
toxic  in  nature. 

The  cause  of  death  in  colubrine  snake  bites  is  chiefly  from  paralysis  of  the  respira- 
tory centers  while  with  the  Pit  Vipers  it  is  chiefly  from  haemorrhages  in  the  vital 
organs.  Antitoxins  have  been  prepared  against  both  viperine  and  colubrine  venoms 
and  these  are  specific,  a  colubrine  antivenin  will  not  be  of  value  against  a  viperine  bite. 
Antivenins  should  be  administered  either  intravenously  or  intramuscularly.  The 
amounts  recommended  for  injections  to  neutralize  a  fatal  dose  of  snake  poison  vary 
from  100  to  300  c.c.  of  the  antivenin  serum.  There  is  no  accurate  standardization. 


NOTES  ON  ANIMAL  PARASITOLOGY. 


NOTES  ON  ANIMAL  PARASITOLOGY. 


NOTES  ON  ANIMAL  PARASITOLOGY. 


NOTES  ON  ANIMAL  PARASITOLOGY. 


PART  IV. 

CLINICAL  BACTERIOLOGY  AND  ANIMAL  PARA- 

SITOLOGY  OF  THE  VARIOUS  BODY 

FLUIDS  AND  ORGANS 


CHAPTER  XXIII. 
DIAGNOSIS  OF  INFECTIONS  OF  THE  OCULAR  REGION. 

IT  is  advisable  before  taking  material  for  cultures  or  smears  to 
cleanse  the  nasal  area  of  the  eye-lids,  and  especially  about  the  caruncles, 
with  sterile  salt  solution.  Then,  by  gently  pressing  on  the  lids,  we  may 
be  able  to  get  pure  cultures  of  the  organism  causing  the  infection. 
Normally,  we  may  find  in  the  region  of  the  caruncles  various  skin 
organisms,  especially  staphylococci,  giving  white  colonies. 

The  xerosis  bacillus  and  white  staphylococci  may  be  considered  normal  findings 
in  the  conjunctival  sac.  Streptococci  and  pneumococci  have  also  been  reported 
from  apparently  normal  conjunctival  secretions. 

A  small  particle  of  sterile  cotton,  wound  on  a  toothpick,  with  the  aid  of  a  sterile 
forceps,  makes  an  excellent  swab  for  obtaining  material  for  smears;  the  same  may 
first  be  drawn  over  an  agar  surface  in  a  Petri  dish  in  a  series  of  parallel  lines  of  in- 
oculation before  making  the  smears  on  slide  or  cover-glass. 

When  there  is  considerable  discharge,  a  capillary  pipette,  with  a  rubber  bulb, 
may  be  used  to  draw  up  sufficient  material  for  cultures  and  smears.  Be  sure  to 
round  off  the  end  of  the  pipette  in  the  flame  and  not  to  use  a  very  fine  capillary 
tube. 

In  conjunctival  cultures,  plates  of  glycerine  agar  or  agar  plates  smeared  with 
blood  are  to  be  preferred,  as  the  gonococcus  and  Koch- Weeks  bacillus  will  only 
grow  on  blood  or  hydrocele  agar.  The  diphtheria  and  xerosis  bacilli  grow  well  on 
glycerine  agar. 

In  addition  to  the  white  staphylococcus,  the  streptococcus  may  be  present 
when  inflammation  of  the  nasal  duct  exists. 

The  Streptococcus  is  at  times  responsible  for  a  pseudo-membranous  conjunctivi- 
tis. The  Staphylococcus  is  as  a  rule  the  cause  of  phlyctenular  conjunctivitis. 

325 


326  INFECTIONS    OF    THE    OCULAR   REGION 

The  pneumococcus  is  a  fairly  common  cause  of  serpiginous  corneal  ulcerations. 
Active  treatment  is  necessary. 

It  is  now  recognized  as  advisable  to  make  an  examination  for  the 
pneumococcus  before  perfoiming  operations  on  the  eye  as  serious 
results  may  follow  if  the  pneumococcus  be  present.  It  is  the  organism 
frequently  found  in  dacryocystitis  and,  in  the  case  of  traumatism,  may 
bring  about  panophthalmitis.  . 

Corneal  ulcerations  are  not  apt  to  appear  even  with  a  pneumococcal  conjunc- 
tivitis unless  there  be  an  injury  of  the  epithelium. 

The  B.  xerosis  is  possibly  a  harmless  organism  and  must  not  be  accepted  as  ex- 
plaining an  infection  unless  other  factors  have  been  eliminated.  The  true  diph- 
theria bacillus,  which  the  xerosis  so  much  resembles,  may  cause  a  pseudomembran- 
ous  inflammation. 

The  B.  pyocyaneus  may  cause  severe  purulent  keratitis  as  well  as  conjunctivitis. 
The  pyocyaneus  toxin  appears  to  be  a  factor. 

The  gonococcus  and  the  Koch-Weeks  bacillus  are  usually  responsible  for  the 
very  acute  cases  of  conjunctivitis.  Both  these  organisms  are  characteristically 
intracellular  and  are  Gram  negative. 

Conjunctivitis  in  the  course  of  epidemic  cerebrospinal  meningitis  has  been 
found  to  be  due  to  the  meningococcus. 

The  diplobacillus  of  Morax  and  Axenfeld  is  more  common  in  chronic,  rather 
dry  affections  of  the  conjunctiva,  chiefly  involving  the  internal  angle  and  showing 
a  morning  accumulation  of  the  secretion.  The  bacilli  are  found  in  twos,  more 
rarely  in  short  chains.  They  are  generally  free  but  may  be  found  in  phagocytic 
cells.  They  resemble  Friedlander's  bacillus  morphologically  but  do  not  have 
capsules. 

In  cases  of  ozena  with  involvement  of  the  nasal  ducts  Friedlander's  bacillus 
may  be  found. 

Even  in  cases  without  ozena,  capsulated,  Gram  negative  bacilli  of  the  Fried- 
lander  group  have  been  frequently  reported  in  conjunctival  inflammation  and  in 
dacryocystitis  as  well. 

The  nodules  of  the  eye-brows  give  the  most  convenient  area  to  take 
material  from  in  the  diagnosis  of  leprosyf  either  the  fluid  expressed 
after  scraping  or  a  piece  of  tissue  cut  into  sections.  Conjunctival  ul- 
ceration  in  leprosy  may  show  abundant  bacilli  as  is  also  true  of  corneal 
ulceration. 

Ordinarily  it  is  impossible  to  find  tubercle  bacilli  in  tuberculous  conjunctival 
discharges. 

The  discharge  from  a  tuberculous  dacryocystitis  may  show  them  satisfactorily. 
Animal  inoculation  is  preferable  in  the  diagnosis  of  ocular  T.  B.  The  pneumo- 
coccus is,  however,  the  most  important  organism  in  dacryocystitis — rarely  the  B. 
coli. 


OCULAR   INFECTIONS  327 

In  a  gonorrhceal  ophthalmia  the  secretion  is  much  more  abundant  and  there  is 
an  absence  of  contaminating  organisms,  the  reverse  of  infection  with  the  confusing 
M.  catarrhalis.  As  a  matter  of  fact,  large  numbers  of  M.  catarrhalis  may  be  present 
in  the  conjunctival  secretion  with  only  slight  irritation  being  observable. 

In  keratomycosis  the  cause  has  been  ascribed  to  Aspergillus  fumigatus. 

Certain  fungi  of  the  genus  Microsporum  have  been  thought  to  be  the  cause  of 
trachoma,  as  have  also  certain  bacillary  forms.  One  should  be  very  conservative 
about  reporting  fungi  in  smears  or  cultures  of  external  surfaces. 

The  larval  stage  of  Taenia  solium  (Cysticercus  cellulosae)  has  a  predilection  for 
eye  as  well  as  brain.  It  is  usually  situated  beneath  the  retina. 

The  question  as  to  the  nature  of  the  so-called  ophthalmic  flukes  is  taken  up  under 
trematodes.  Echinococcus  cysts  have  been  reported  in  the  orbit. 

The  adult  Filaria  loa  tends  at  times  to  appear  under  the  conjunctiva  or  in  the 
subcutaneous  tissue  of  the  eye-lids. 

Fly  larvae  have  been  reported  from  the  conjunctival  sacs  in  the  helpless  sick. 

Demodex  may  cause  an  obstinate  blepharitis. 

Prowazek  has  thought  that  certain  fine  dots  within  the  cytoplasm  of  epithelial 
cells,  which  stain  best  by  Giemsa's  method  and  which  he  considered  protozoal  in 
nature,  were  the  cause  of  trachoma. 


CHAPTER  XXIV. 

DIAGNOSIS   OF  INFECTIONS   OF  THE  NASAL  AND  AURAL 

CAVITIES. 

IN  taking  material  from  the  nasal  cavities,  for  bacteriological 
examination,  it  is  well  to  wash  about  the  alae  with  sterile  water  and 
then  have  the  patient  blow  his  nose  on  a  piece  of  sterile  gauze  and  take 
the  material  for  culture  or  smear  from  this.  If  the  material  is  purulent 
and  located  at  some  ulcerating  spot,  it  is  best  to  use  a  speculum,  and 
either  touch  the  spot  with  a  sterile  swab  or  use  a  capillary  bulb  pipette 
with  a  slight  bend  at  the  end. 

Normally,  we  find  only  white  staphylococcus  colonies  and  colonies  of  short-chain 
streptococci.  The  M.  tetragenus,  B.  xerosis,  and  Hoffman's  bacillus  are  also 
occasionally  found. 

In  some  cases  of  ozena  we  may  find  an  organism  of  the  Friedlander  type  in  pure 
culture. 

Biscuit-shaped  diplococci,  both  Gram  negative  and  positive,  are  to  be  found 
either  normally  or  in  cases  of  coryza.  M.  catarrhalis  has  probably  been  frequently 
reported  as  the  meningococcus.  Still,  the  meningococcus  has  been  found  in  the 
nasal  secretions  of  patients  with  cerebrospinal  meningitis.  B.  influenzas  and  the 
pneumococcus  have  also  been  frequently  found  in  cultures  from  the  nasal  secretions. 

Diphtheria  involving  the  nasal  cavity  must  always  be  kept  in  mind,  and  in 
quarantine  investigations  the  examination  of  the  nasal  secretions  culturally  should 
be  a  part  of  the  routine. 

The  tubercle  bacillus  may  be  found  in  nasal  ulcerations;  it  is,  however,  only 
present  in  exceedingly  small  numbers.  On  the  other  hand,  one  of  the  best  diagnostic 
procedures  in  leprosy  is  to  examine  smears  from  nasal  mucous  membrane  for  the 
B.  leprae.  In  such  ulcerations  the  bacilli  are  found  in  the  greatest  profusion.  Rarely 
glanders  may  cause  ulcerations. 

B.  proteus  is  frequently  responsible  for  the  production  of  foul  odors  in  nasal 
discharges  but  does  not  seem  to  produce  inflammatory  conditions  of  the  nasal 
mucosa.  It  simply  decomposes  the  discharges.  Various  fungi  have  been  reported 
from  the  nose,  but  in  such  a  region  the  strictest  conservatism  in  reporting  should  be 
observed. 

Recently  sporozoa  have  been  reported  in  a  case  of  nasal  polyp.  (Rhinosporidium.) 

So  many  degenerative  changes  in  epithelial  cells  resemble  protozoal  forms  that 
such  findings  require  ample  confirmation. 

The  larval  form  of  Linguatula  rhinaria  is  a  rare  parasite  of  the  nasal  cavities; 
it  is  not  infrequent,  however,  in  the  nostrils  of  dogs. 

328 


EAR  INFECTIONS  329 


Various  fly  larvae  are  far  more  common,  and  the  "  screw- worm," 
the  larva  of  the  Chrysomyia  macellaria,  is  common  in  certain  parts 
of  tropical  America,  and  may  by  its  burrowing  effects  cause  fatal  results. 

The  larvae  of  Sarcophaga  have  in  particular  been  found  in  the  nasal  cavities  of 
children.  Myriapods,  while  of  very  little  importance  elsewhere,  have  been  reported 
more  than  thirty  times  from  the  nasal  fossae. 

In  a  study  of  the  bacteriology  of  otitis  media,  in  277  cases,  Libman 
and  Celler  found  streptococci  present  alone  in  81%,  streptococcus 
mucosus  in  10%  and  the  pneumococcus  in  8%;  staphyococci,  B.  pyo- 
cyaneus  and  B.  proteus  have  also  been  found.  Mixed  infections  are 
common. 

Streptococci  are  the  organisms  which  most  often  cause  sinus  thrombosis  and  brain 
abscess.  The  influenza  bacillus  has  been  reported  as  a  cause  of  acute  otitis  media. 

Nonvirulent  diphtheroid  bacilli  are  not  infrequently  obtained  in  cultures  from 
ear  discharges. 

Other  organisms  which  have  been  isolated  from  middle  ear  or  mastoid  discharges 
are  B.  coli,  M.  catarrhalis,  M.  tetragenus  and  Friedlander's  bacillus. 

B.  typhosus  may  be  found  in  middle-ear  discharges  of  persons  who  have  had  an 
attack  of  typhoid  fever. 

The  middle  ear  is  normally  free  of  bacteria,  but  in  affections  of  the  throat,  as 
with  streptococci,  pneumococci,  and  diphtheria  bacilli,  these  organisms  may  infect 
it  by  way  of  the  Eustachian  tube. 

The  moulds  are  of  greater  importance  in  affections  of  the  external  auditory  canal 
than  the  bacteria.  The  cerumen  seems  to  make  a  good  culture  medium  so  that 
various  species  of  Aspergillus,  Mucor,  etc.,  develop  and  close  the  canal.  These 
infections  are  often  introduced  by  the  patient's  finger.  Various  mites  and  fly  larvae 
have  been  reported  from  the  ear. 


CHAPTER  XXV. 
EXAMINATION  OF  BUCCAL  AND  PHARYNGEAL  MATERIAL. 

IN  a  preparation  made  from  material  taken  by  a  sterile  swab  from 
the  region  of  the  normal  buccal  and  pharyngeal  cavities  and  stained  by 
Gram's  method  we  are  struck  by  the  variety  of  organisms  present. 

Gram  positive  and  Gram  negative  staphylococci  are  present,  as  are  also  strepto- 
cocci, pneumococci,  leptothrix  forms,  and  very  probably  yeasts  and  sarcinae  types 
with  many  Gram  negative  bacilli.  If  pseudo-diphtheria  organisms  are  present, 
we  have  these  showing  a  Gram  positive  reaction.  If  this  material  is  smeared  on 
agar  plates  and  cultured  at  37°  C.,  we  are  struck  by  the  fact  that  the  colonies  on 
the  plates  may  be  exclusively  staphylococcal  and  streptococcal. 


FIG.  97. — Vincent's  angina.     Spirochaeta  vincenti.     (Coplin.} 

•* 

It  is  very  difficult,  if  not  impossible,  to  distinguish  a  pneumococcus  colony  from 
•a  streptococcus  one  on  a  plate  culture.  The  presence  or  absence,  however,  of  the 
pneumococcus  is  distinctly  shown  in  the  Gram-stained  smear,  either  by  its  lance- 
shaped  morphology  or  the  presence  of  a  capsule.  It  has  been  my  experience  that 
smears  from  about  15%  of  normal  individuals  show  capsulated  pneumococci. 

In  diphtheria  examinations  we  rely  chiefly  on  the  cultural  findings  on  Lb'ffler's 
serum.  Where  the  process  is  streptococcal  or  due  to  the  organisms  associated  with 
Vincent's  angina,  the  immediate  examination  of  a  smear  from  the  suspected  spot 
or  area  gives  greater  diagnostic  information.  The  streptococcus  being  so  abundant 
in  cultures  from  normal  throats,  it  is  difficult  to  determine  its  significance  in  a  cul- 

330 


THROAT   INFECTIONS  331 

ture;  abundance  of  streptococci  in  a  smear  from  an  ulceration  or  bit  of  membrane, 
however,  is  of  etiological  import. 

By  staining  with  Neisser's  method  it  is  possible  to  make  an  imme- 
diate diagnosis  of  diphtheria  from  a  smear  from  a  piece  of  membrane 
in  about  25  %  of  cases.  It  is  well,  however,  to  always  culture  such 
material.  The  toluidin  blue  stain  of  Ponder  is  the  best  stain  for 
diphtheria. 

Material  from  the  throat  is  ordinarily  best  obtained  with  a  sterile  copper-wire 
cotton-pledget  swab.  The  platinum  loop  usually  bends  too  easily.  A  sterile  for- 
ceps may  be  more  convenient  for  obtaining  particles  of  membrane.  It  is  believed 
that  ulcerative  conditions  of  the  throat,  associated  with  the  presence  of  the  large 
fusiform  bacillus  and  delicate  spirillum,  which  make  the  picture  of  Vincent's  angina, 
are  more  common  than  is  usually  so  considered. 

In  Giemsa  stained  smears  from  the  dirty  membrane  covering  the  ulcerated  area 
of  Vincent's  angina  there  are  usually  two  types  of  the  fusiform  bacillus  to  be  seen; 
one  rather  slender,  pale  blue  with  maroon  dots  at  either  end,  the  other  rather  thicker 
and  of  a  uniform  maroon  staining.  The  spirilla  are  from  10  to  18  microns  long  and 
the  fusiform  bacilli  from  5  to  7  microns. 

As  a  rule,  only  cultures  on  serum  are  made  and  very  rarely  direct  smears.  If  a 
smear  were  always  made  and  stained  by  Gram's  method  (with  a  contrast  stain  of 
dilute  carbol  fuchsin)  at  the  same  time  the  culture  was  made,  it  is  probable  that 
much  information  of  value  would  be  obtained. 

The  B.  fusiformis  is  an  anaerobe  which  gives  a  fetid  odor  but  culturally  has  no 
distinct  characteristics.  The  spirillum  has  not  been  cultivated.  It  has  been 
thought  that  the  bacillus  and  spirillum  are  different  stages  of  the  same  organism. 
At  times  aggregations  of  the  fusiform  bacillus  give  the  appearance  of  branching  so 
characteristic  of  diphtheria  organisms.  Being  Gram  negative,  however,  the  differ- 
entiation is  easily  made — the  B.  diphtherias  being  Gram  positive.  Again  the 
attenuated  ends  of  the  fusiform  bacillus  are  diagnostic. 

Direct  smears  are  the  procedure  of  choice  in  streptococcal  and 
pneumococcal  anginas  as  well  as  in  Vincent's  angina. 

Unless  very  familiar  with  the  morphology  of  Treponema  pallidum  and  using 
Giemsa's  staining  procedure,  we  should  be  very  conservative  in  reporting  such  an 
organism  from  suspected  syphilitic  ulcerations  of  the  throat. 

The  thrush  fungus  (Endomyces  albicans)  may  be  easily  demonstrated  in  a  Gram- 
stained  specimen  as  violet  mycelial  structures. 

Yeasts  due  to  food  particles  are  not  infrequently  observed  in  smears  and  cultures 
from  the  mouth. 

Actinomycosis  may  develop  about  a  carious  tooth  and  the  finding  of  the  ray 
fungus  in  the  granules  from  the  pus  may  give  the  diagnosis. 

Amoebae  and  flagellates  have  been  reported  from  the  mouth.  Also  in  the  re- 
markable disease  "halzoun,"  flukes  have  been  found  to  be  the  cause  of  the  asphyxia. 


332     EXAMINATION   OF  BUCCAL  AND   PHARYNGEAL   MATERIAL 

In  the  tropics,  round  worms  may  be  vomited  up  and,  lodging  in  the  pharynx, 
may  have  to  be  extracted. 

During  the  campaign  of  Napoleon  in  Egypt  many  cases  of  leech  involvement  of 
the  nasal  and  buccal  cavities  were  noted.  The  parasite  was  the  Limnatis  nilotica 
which  gained  access  to  the  upper  pharynx  through  drinking  water  from  springs  and 
pools.  Many  such  cases  continue  to  be  reported  from  the  Mediterranean  basin. 


CHAPTER  XXVI. 
EXAMINATION  OF  SPUTUM. 

FREQUENTLY  the  material  submitted  for  examination  as  sputum  is 
simply  buccal  or  pharyngeal  secretion,  or  more  probably  secretion 
from  the  nasopharynx,  which  has  been  secured  by  hawking.  It  should 
always  be  insisted  upon  that  the  sputum  be  raised  by  a  true  pulmonary 
coughing  act,  and  not  expelled  with  the  hacking  cough  so  frequently 
associated  with  an  elongated  uvula.  When  there  is  an  effort  to  deceive, 
some  information  may  be  obtained  from  the  watery,  stringy,  mucoid 
character  of  the  buccopharyngeal  material  and  also  fiom  the  presence 
of  mosaic-like  groups  of  flat  epithelial  cells  (often  packed  with  bacteria). 
The  pulmonary  secretion  is  either  frothy  mucus  or  mucopurulent  mate- 
rial, and  if  the  cells  are  alveolar  they  greatly  resemble  the  plasma  cells. 
At  times  these  cells  may  contain  blood  pigment  granules  (heart-disease 
cells). 

In  the  microscopic  examination  a  small,  cheesy  particle,  the  size  of  a  pin  head, 
should  be  selected.  This  should  be  flattened  out  in  a  thin  layer  between 
the  slide  and  cover-glass  and  should  be  examined  for  elastic  tissue,  heart-disease 
cells,  eggs  of  animal  parasites,  amoebae,  and  fungi.  Echinococcus  booklets,  Cursch- 
man  spirals  besprinkled  with  Charcot-Leyden  crystals,  and  haematoidin  and  fatty 
acid  crystals  may  also  be  observed. 

Curschman  spirals  indicate  bronchial  as  against  cardiac  or  uremic  asthma. 
Charcot-Leyden  crystals  have  no  special  significance,  except  in  certain  tropical 
diseases  when  these  crystals  often  are  present  in  paragonomiasis  sputum  and  in 
the  pus  of  amoebic  liver  abscesses  discharging  by  way  of  the  lungs. 

It  may  facilitate  the  examination  of  the  sputum  for  elastic  tissue  and  actinomy- 
cosis  and  other  fungi  to  add  10%  sodium  hydrate  to  the  preparation. 

To  make  smears  for  staining,  the  sputum  should  be  poured  on  a  flat  surface, 
preferably  a  Petri  dish,  and  a  bit  of  mucopurulent  material  selected  with  forceps.  A 
dark  back-ground  facilitates  picking  out  the  particle.  A  toothpick  is  well  adapted 
to  smearing  out  such  material  on  a  slide.  After  using  the  toothpick  it  can  be  burned. 
When  dry,  the  smear  is  best  fixed  by  pouring  a  few  drops  of  alcohol  on  the  slide, 
allowing  this  to  run  over  the  surface,  and  then,  after  dashing  off  the  excess  of  alcohol, 
to  ignite  that  remaining  on  the  film  in  the  flame  and  allow  to  burn  out. 

A  mark  with  a  grease  pencil,  about  1/2  inch  from  the  end,  gives  a  convenient 
surface  to  hold  with  the  forceps  and  also  prevents  the  stain  subsequently  used  from 
running  over  the  entire  surface.  A  piece  of  glass  tubing  about  12  inches  long  bent 

333 


334  EXAMINATION   OF   SPUTUM 

into  a  narrow  V  shape  makes  a  very  satisfactory  rest  for  the  slide  in  staining  and  is 
convenient  for  the  steaming  of  staining  solution  over  the  flame. 

Sputum  should  as  a  routine  measure  be  stained  by  the  Ziehl-Neelson  method  and 
by  Gram's  method. 

In  examining  for  tubercle  bacilli  it  may  be  necessary  to  employ  some  method  for 
concentrating  the  bacterial  content  of  the  sputum  prior  to  making  the  smear.  A  very 
satisfactory  method  is  that  of  Miihlhauser-Czaplewski.  Shake  up  the  sputum  with 
four  to  eight  times  its  volume  of  1/4%  solution  of  sodium  hydrate  in  a  stoppered 
bottle.  When  the  mixture  has  become  a  smooth,  mucilaginous-looking  fluid,  add  a 
few  drops  of  phenolphthalein  solution  and  bring  the  pink  mixture  to  a  boil. 

Then  add  drop  by  drop  a  2%  solution  of  acetic  acid,  stirring  constantly,  until 
the  pink  color  is  just  discharged.  If  the  least  excess  of  acid  is  added  over  that  just 
sufficient  to  cause  the  pink  color  to  disappear,  mucin  will  be  precipitated.  Now  pour 
this  mixture  into  a  centrifuge  tube  and  smear  the  sediment  on  a  slide  and  stain  for 
tubercle  bacilli. 

Tubercle  bacilli  usually  occur  nested  in  clumps  of  sputum.  There- 
fore, when  few  in  number  it  is  only  by  chance  that  they  may  be  found. 
Concentration  methods  aim  to  dissolve  these  clumps  of  sputum  and 
collect,  free  from  mucus,  whatever  bacilli  may  be  present.  There  are 
many  concentration  methods  for  sputum.  One  of  these  has  been 
given  above.  Uhlenhuth's  method  has  some  advantages  over  others 
in  the  solvent  used:  i.  It  breaks  up  the  sputum  very  rapidly;  2.  it 
immediately  dissolves  all  organisms  except  acid-fast  ones;  3.  applied 
in  not  too  concentrated  form  and  for  not  too  long  a  time,  tubercle 
bacilli  are  not  killed,  so  that  by  washing  the  sediment  carefully  by 
several  dilutions  and  centrifugings  we  have  in  the  sediment  viable 
tubercle  bacilli  which  we  may  attempt  to  cultivate  upon  Dorset's  or 
other  suitable  media  with  the  reasonable  hope  that  contaminations  will 
not  choke  them  out  or  prematurely  kill  the  inoculated  guinea-pig;  4. 
it  has  less  effect  upon  the  staining  properties  of  tubercle  bacilli  than 
any  other  material  used  in  concentration  methods. 

To  make  this  solvent  (antiformin)  take  double  the  quantity  of  chlorinated  lime 
and  sodium  carbonate  required  by  the  U.  S.  Pharmacopoeia  and  prepare  according 
to  U.  S.  P.  directions.  To  the  finished  liquor  sodae  chlorinatae  (Labarraque's  solu- 
tion) add  7  1/2%  of  sodium  hydrate. 

The  Liquor  sodae  chlorinatae  of  the  Br.  P.  is  slightly  stronger  and  some  English 
authorities  recommend  a  mixture  of  equal  parts  of  this  Labarraque's  solution  and 
r5%  sodium  hydrate  solution.  As  a  rule  one  part  of  antiformin  to  five  parts  of 
sputum  is  sufficient.  Very  tenacious  sputum  may  require  one  part  to  four  parts 
of  sputum.  If  more  antiformin  is  used  the  specific  gravity  is  too  much  increased 
and  the  bacilli  are  damaged.  The  fluidification  is  hastened  at  incubator 
temperature. 

To  five  parts  of  sputum  add  one  part  of  antiformin,  shake  well  and  place  in 


TUBERCULOUS  SPUTUM  335 

incubator  for  one  hour.  To  10  c.c.  of  the  homogeneous  mixture  add  1.5  c.c.  of  a 
solution  made  up  of  one  part  chloroform  and  nine  parts  alcohol.  Shake  violently 
and  centrifuge  for  15  minutes.  Mix  the  sediment  wit)/  egg  albumin,  smear  out  and 
stain. 

When  it  is  desired  to  culture  the  tubercle  bacilli  mix  20  c.c.  of  sputum  with  65  c.c. 
sterile  water  and  add  15  c.c.  antiformin.  Stir  the  mixture  with  a  glass  rod.  After 
30  minutes  to  two  hours  we  should  have  a  homogeneous  mixture.  Centrifuge  for 
15  minutes  or  longer,  wash  the  sediment  twice  with  sterile  salt  solution  and  smear  out 
the  well-washed  sediment  over  serum  or  glycerine  egg  slants.  The  tubes  should  be 
covered  with  black  paper  and  the  plugs  paraffined.  It  must  be  remembered  that  for 
culturing  tubercle  bacilli  we  must  protect  the  growth  from  sunlight  as  this  will  kill 
the  organism.  If  fluid  culture  media  are  inoculated  the  transferred  material  should 
be  deposited  on  the  surface.  Should  the  particle  sink  growth  will  not  occur. 

Sputum  smears  stained  by  some  Romanowsky  method  or  by  the  haematoxylin- 
eosin  stain  are  best  adapted  for  the  study  of  various  cells,  and  in  particular  of  the 
eosinophile  cells  so  characteristic  of  bronchial  asthma.  In  sputum  from  cancer  of 
the  lungs  the  large  vacuolated  cells  may  be  found. 

When  examining  the  sputum  of  the  bronchopneumonia  of  influenza  the  formol 
fuchsin  gives  the  best  results.  The  influenza  bacilli  are  found  in  little  masses,  fre- 
quently grouped  about  small  collections  of  M.  tetragenus.  The  cocci  stain  a  rich 
purplish-red,  while  the  small  influenza  bacilli  take  on  a  light  pink  color. 

T.  B.  sputum  showing  a  mixed  infection  with  streptococci  or  pneumococci  or  with 
the  influenza  bacillus  makes  for  a  bad  prognosis.  M.  tetragenus,  which  often  is 
present  when  cavities  exist,  does  not  seem  to  be  so  unfavorable  prognostically. 

Red  cells  show  up  well  in  specimens  stained  by  the  Romanowsky  method;  if 
rouleaux  formation  is  marked,  it  may  indicate  pulmonary  infarction. 

In  culturing  sputum  a  mucopurulent  mass  should  be  washed  in 
sterile  water  and  should  then  be  dropped  into  a  tube  of  sterile  bouillon. 
With  a  sterile  swab  it  should  be  emulsified  and  successive  streaks  made 
along  the  surface  of  an  agar  or  glycerine  agar  plate.  In  obtaining  cul- 
tures from  influenza  sputum,  first  smear  the  material  thoroughly  over 
a  blood-serum  slant;  then  inoculate,  by  thorough  smearing  over  the 
surface  of  successive  blood-stieaked  agar  slants,  the  material  on  the 
surface  of  the  blood-serum  slant.  The  platinum  loop  should  be  trans- 
ferred from  one  slant  to  another  without  recharging.  The  influenza 
bacillus  seems  to  grow  better  if  the  blood-streaked  agar  slants  are 
prepared  just  before  inoculating  with  the  sputum.  All  that  is  necessary 
is  to  sterilize  an  ear,  puncture  and  take  up  the  exuding  blood  with  a 
large  loop.  Cultures  for  tubercule  bacilli  are  impracticable  except  with 
antiformin.  A  guinea-pig  should  be  inoculated. 

The  blood-stained  watery  sputum  of  plague  pneumonia  should  be  cultured  on 


336  EXAMINATION   OF   SPUTUM 

plates  of  plain  agar  and  3  %  salt  agar  at  the  same  time.  An  ordinary  smear  stained 
with  carbol  thionin,  however,  practically  makes  a  diagnosis. 

Pneumococci,  M.  catarrhalis,  and  Friedlander's  bacillus  in  sputum  are  best 
demonstrated  by  Gram's  method  of  staining. 

The  distinct  capsule  staining  of  the  pneumococci  in  a  Gram  preparation  of 
sputum  from  a  suspected  case  of  pneumonia  is  of  value  in  diagnosis. 

The  finding  of  the  ray  fungus  (D.  bovis)  in  sputum  gives  the  diagnosis  of  actino- 
mycosis.  Streptothrix  infections  of  lungs  have  been  confused  with  tuberculosis. 

Moulds,  especially  Aspergilli,  may  be  found  in  sputum.  Species  of  Mucor, 
Cryptococcus,  and  Endomyces  have  also  been  reported. 

Amoebae  from  liver  abscess  rupturing  into  the  lung  may  be  found.  Very  impor- 
tant pulmonary  infections  are  those  with  Paragonimus  westermanii.  This  is  recog- 
nized by  the  presence  of  operculated  eggs  in  the  sputum. 

A  fluke,  F.  gigantea,  was  once  found  in  sputum. 

Hydatid  cysts,  either  of  the  lung  or  of  the  liver,  rupturing  into  the  lung,  may  be 
recognized  by  the  presence  of  echinococcus  hooklets.  The  material  is  bile-stained 
if  from  the  liver. 

Strongylus  apri  has  been  reported  once  from  the  lungs  and  embryos  might  be 
found  in  the  sputum.  In  pulmonary  bilharziosis  Schistosoma  eggs  may  be  found  in 
the  sputum. 

The  test  for  ALBUMEN  IN  THE  SPUTUM  is  of  value  in  the  diagnosis  of  pulmonary 
tuberculosis. 

About  10  c.c.  of  fresh  sputum  as  pure  as  possible  from  saliva  is  mixed  with  an 
equal  quantity  of  water  and  2  c.c.  of  a  3%  solution  of  acetic  acid  to  remove  mucin. 
After  filtering  the  filtrate  is  tested  for  albumin.  The  test  is  obtained  also  in 
pneumonia  and  pleurisy  with  effusion. 


CHAPTER  XXVII. 


THE  URINE. 

MATERIAL  for  staining  is  best  obtained  by  centifuging  the  urine, 
then  pouring  off  the  supernatant  urine,  then  draining  the  mouth  of 
the  centrifuge  tube  against  a  piece  of  filter-paper  so  that  we  have  only 
the  pus  sediment  to  finally  remove  with  a  capillary  bulb  pipette  and 
make  smears. 

The  addition  of  a  loopful  of  egg  albumen  or  blood  serum  to  about  twice  that 
amount  of  urinary  sediment  gives  better  results.  (See  under  Staining  Methods.) 


Orange      'Yellow  Green 

B    C  D  c 


Oxy  haemoglobin 


MeJkaemo<globin  -  alkaline 


Mefhaemoglobin  —  -faintly  acid 


Corborv   monoxide  haemoqlobm 


haemoglobin 

FIG.  98. — i,  Various  absorption  bands  of  spectrum;  2,  crystals  of  glucosazone 
(Phenylhydrazine sugar  test);  3,  Cammidge  crystals  (interacinar  type  of  pancreati- 
tis); 4,  Cammidge  crystals  (interlobular  type  of  pancreatitis). 

The  smear  may  be  stained  directly  by  Wright's  method  or  after  fixing  by  heat 
with  Gram's  stain,  T.  B.  stain,  or  haematoxylin  and  eosin.  The  latter  is  the  best  for 
the  staining  of  epithelial  cells  and  animal  parasites;  the  Gram  method  for  bacteria. 

It  is  frequently  difficult  to  distinguish  the  spores  of  moulds  from  red  blood-cells 
except  by  measurement  and  staining  reactions.  Spores  of  moulds  rarely  exceed 
five  microns. 


22 


337 


338 


THE    URINE 


It  is  difficult  to  determine  the  presence  of  blood  in  urine  in  higher  dilution  than 
i  to  300  with  the  spectroscope.  The  ordinary  occult  blood  test  will  show  it  in  much 
higher  dilution. 

To  secure  urine  for  bacteriological  examination  catheterization  is  rarely  necessary 
in  men — in  the  case  of  women  it  is  the  proper  method. 

The  glans  penis  and  meatus  should  be  thoroughly  washed  with  soap  and  water, 
after  which  dilute  alcohol  (50%)  should  be  used.  The  greater  part  of  the  urine 
first  passed  should  be  rejected  and  only  the  last  portion  passed  should  be  caught  in 
a  sterile  receptacle.  A  drop  of  this  urine  may  be  either  streaked  over  the  surface 
of  an  agar  or  a  lactose  litmus  agar  plate,  or  so  treated  after  being  first  diluted  in  a 
tube  of  sterile  bouillon. 

The  lactose  litmus  agar  medium  is  very  useful  in  distinguishing  typhoid  or  para- 


O       o 

On* 

oW    |. 

XJcwx 


FIG.  99. — Starches  and  fibers  found  in  urine. 

typhoid  colonies  (blue)  from  colon,  and  streptococcus  or  staphylococcus  colonies 
(pink).  The  urine  may  be  added  to  tubes  of  melted  agar  and  then  poured. 

The  most  satisfactory  procedure  is  to  deposit  one  drop  on  a  poured  plate  and  five 
drops  on  a  second  plate.  The  surface  is  smeared  over  with  a  bent  glass  rod  first 
smearing  out  the  single  drop  and  then  going  to  the  second  plate  without  a  second 
sterilization.  Neutral  glycerine  agar  or  blood  agar  is  desirable  for  such  organisms 
as  pneumococci  or  streptococci  and,  for  the  gonococcus,  Thalman's  medium  smeared 
over  with  a  few  drops  of  human  serum. 

Cystitis  from  a  colon  infection  gives  an  acid  urine;  that  caused  by  Proteus 
vulgaris  an  alkaline  urine. 


BACTERIAL   INFECTIONS    OF    URINE  339 

The  old  designation  B.  termo  so  often  employed  in  connection  with  the  bacteri- 
ology of  the  urine  in  older  works  applied  to  the  proteus  group  and  M.  ureae  to  ordi- 
nary staphylococci. 

The  bacillus  of  typhoid  and  the  micrococcus  of  Malta  fever  are  also 
found  in  the  urine.  This  elimination  in  urine  of  bacilli  by  typhoid 
carriers  is  of  great  importance  in  the  spread  of  the  disease. 

While  the  smegma  bacillus  in  urine  may  be  differentiated  from  the  tubercle 
bacillus  by  the  former  losing  its  red  color,  by  prolonged  decolorization  with  acid 
alcohol,  yet  it  is  chiefly  by  the  subcutaneous  inoculation  of  the  guinea-pig  that  we 
should  diagnose  genito-urinary  tuberculosis.  Inject  the  sediment  after  centrifuging. 

The  method  recommended  by  Gasis  which  depends  on  the  alkali  fast  properties 
of  the  T.  B.  has  not  given  me  satisfactory  results. 

Gonococci  are  reported  from  Gram-stained  smears. 

To  culture  gonococcus  material  the  transfer  to  culture  media  should  be  made 
almost  immediately  after  obtaining  the  material  from  the  patient.  M.  catarrhalis 
is  a  rare  finding. 

Staphylococcus  and  Streptococcus  infections  about  the  mouth  as  well  as  such 
infections  in  heart  or  joint  may  show  the  presence  of  the  causative  organisms  in 
the  urine.  At  times  bacterial  infections  of  the  kidney  may  give  symptoms  of  renal 
stone. 

As  it  is  much  easier  to  culture  urine  than  blood  a  bacteriological  examination  of 
the  urine  may  give  us  the  desired  information  and  the  organism  for  the  autogenous 
vaccine.  Salt  mouth  bottles  with  cotton  plugs,  when  sterilized,  make  cheap  and 
satisfactory  containers.  The  urine  should  be  plated  out  as  soon  as  possible  after 
its  passage.  As  a  rule  when  organisms  are  present  in  the  urine  they  are  in  such 
numbers  that  the  question  of  contamination  rarely  arises. 

Yeasts  and  moulds  frequently  contaminate  urine,  especially  diabetic  urine, 
after  it  has  been  passed.  Amoebae  and  flagellates  (Trichomonas  vaginalis  in  females) 
may  be  found  in  urine. 

Eggs  of  Schistosomum  haematobium  (bilharziosis)  are  important  diagnostic 
findings;  these  are  terminal-spined.  Those  of  rectal  bilharziosis  are,  as  a  rule, 
lateral-spined. 

In  chylous  urine  the  filarial  embryos  may  be  found.  This  examination  is  facili- 
tated by  centrifugalization. 

The  eggs  of  the  E.  gigas  may  be  recognized  in  urinary  sediment  by  their  pitted 
appearance. 

The  vinegar  eel  may  be  found  in  the  urine  of  females  who  have  used  vaginal 
douches  of  vinegar. 

Echinococcus  hooklets,  scolices,  or  laminated  membrane  have  been  found  in  the 
urine. 

The  larval  dibothriocephalid,  Sparganum  mansoni,  has  been  reported  three  times 
in  urine  (urethra). 

Oxyuris  from  the  vagina  may  be  found  in  urine. 

Various  mites  may  be  found  in  urinary  sediment  as  the  result  of  lack  of  care  in 
the  washing  of  the  receptacle  and  are  entirely  accidental. 


340 


THE    URINE 


Unless  having  the  characteristics  of  the  itch  mite  and  in  a  person  showing  scabies 
lesions  about  the  genital  organs  the  diagnosis  of  the  mite  as  A.  Scabiei  should 
not  be  made. 

Crystals  of  biliverdin  may  be  found  in  the  urinary  sediment  in  marked  jaundice. 
They  somewhat  resemble  crystals  of  tyrosin  but  are  brownish  in  color  while  those 
of  tyrosin  are  black.  Furthermore,  it  is  excessively  rare  to  find  crystals  of  leucin 
and  tyrosin  in  the  urinary  sediments,  and  in  such  diseases  as  acute  yellow  atrophy  of 
the  liver,  the  urine  should  be  concentrated  to  one-tenth  its  volume  and  the  residue 
treated  with  alcohol.  The  tyrosin  crystalline  sheaves  and  the  leucin  striated 
globules  crystallize  out  from  the  alcohol. 


I 


s 


FIG.  IOQ. — Epithelium  from  different  areas  of  the  urinary  tract,  a,  Leukocyte 
(for  comparison);  b,  renal  cells;  c,  superficial  pelvic  cells;  d,  deep  pelvic  cells;  e, 
cells  from  calices;  /,  cells  from  ureter;  g,  g,  g,  g,  g,  squamous  epithelium  from  the 
bladder;  h,  h,  neck-of-bladder  cells;  i,  epithelium  from  prostatic  urethra;  k,  urethra! 
cells;  I,  I,  scaly  epithelium;  m,  m' ',  cells  from  seminal  passages;  n,  compound  granule 
cells;  o,  fatty  renal  cell.  (Ogden.} 


URINARY  SEDIMENTS. 

Turbidity  of  the  urine  is  most  often  due  either  to  bacterial  contamination, 
amorphous  urates  (sedimentum  lateritium)  or  phosphates. 

Urates  go  into  solution  upon  heating  and  phosphates  upon  the  addition  of  a  few 
drops  of  acetic  acid. 

In  turbidity  due  to  bacteria  contaminating  the  urine  subsequent  to  its  passage 
it  is  best  to  call  for  another  sample. 

To  preserve  urinary  sediments  formalin  is  the  best  for  casts  and  epithelial  cells 
while  for  general  use  one  may  employ  a  piece  of  camphor  or  the  addition  of  one 
volume  of  saturated  borax  solution  to  four  volumes  of  urine. 


URINARY   CRYSTALS 


341 


Chloroform  does  not  answer  for  sediments  as  it  does  for  urine  to  be  examined 
chemically.  To  take  up  a  sediment  insert  a  pipette  to  the  bottom  of  the  tube  with 
the  opposite  opening  closed  by  a  finger,  then  tease  the  sediment  into  the  pipette 
opening  in  the  centrifuge  tube,  by  manipulating  the  fingers. 


«^*»i.tt 


k  r^l^K   £**_**&  <S3/  ^  \ 


-  e 


FIG.   101. — Deposit  in  acid  fermentation,     a,  Fungus;  b,  amorphous  sodium  urate; 
c,  uric  acid;  d,  calcium  oxalate. 

In  a  urine  of  acid  reaction  we  may  find  the  following  unorganized  sediment: 
I.  Amorphous  sodium  or  potassium  urates.     Usually  yellowish  red.     Heat  and 
alkali  bring  about  solution. 


-i, 


FIG.   102. — Deposit  in  ammoniacal  fermentation,     a,  Acid  ammonium  urate;  b, 
ammonium   magnesium   phosphate;    c,    bacteria. 

II.  Uric  acid.  Whetstone  crystals  of  yellowish-red  color.  Soluble  in  alkalis 
but  not  by  heat.  Abundant  sediment  of  uric  acid  crystals  may  be  due  to  too  great 
concentration  or  too  great  acidity  of  the  urine  rather  than  to  the  so-called  uric  acid 
diathesis. 


342  THE    URINE 

III.  Calcium   oxalate.     Octahedral  crystals    or    dumb-bell  shapes   which   are 
highly  refractile.     Often  due  to  diet  (asparagus,  tomatoes,  spinach,  rhubarb,  etc.). 

IV.  Cystin  occurs  in  six-sided  crystals  which  are  soluble  in  ammonia. 
In  a  urine  of  alkaline  reaction  we  may  expect: 

I.  Triple  phosphates  (NH4  MgPO4).     Usually  in  coffin-lid  or  fern-like  form. 
Easily  soluble  in  acetic  acid. 

II.  Calcium  phosphate  and  calcium  carbonate  which  effervesce  on  the  addition 
of  acid. 

III.  Ammonium  urate.     These  show  as  the  thorn-apple  structures. 

The  presence  of  ammonium  urate,  particularly  if  with  triple  phosphates,  denotes 
bacterial  decomposition  within  the  genito-urinary  tract  provided  the  urine  is  just 


i 

FIG.  103. — Fatty  and  waxy  casts,     a,  Fatty  casts;    b,  waxy  casts. 


(Greene.) 


passed.  Pus  cells  derived  from  the  site  of  inflammation  should  be  present  also. 
While  certain  bacteria  might  possibly  bring  on  chemical  changes  without  giving  rise 
to  inflammation  yet  such  a  possibility  is  so  rare  as  to  be  negligible.  In  the  presence 
of  amorphous  phosphates  one  should  always  think  of  exogenous  sources  as  vegetable 
diet  or  withdrawal  of  proteid  food  before  thinking  of  disordered  metabolism. 

Organized  Sediment. — An  occasional  leukocyte  may  be  found  in  the  urine  of 
healthy  people.  Any  abundance  of  leukocytes  indicates  inflammation  of  genito- 
urinary tract.  Some  workers  count  the  pus  cells  in  urine  by  the  same  technic  used 
for  the  leukocyte  count  of  the  blood.  A  urine  having  100,000  pus  cells  per  c.mm. 
will  give  as  a  result  about  0.1%  albumin. 

Leukocytes  are  found  in  abundance  at  times  in  the  urine  of  women  without 


URINARY   CASTS  343 

pathological  significance.  Red  blood  cells  usually  show  as  pale  doubly  ringed 
bodies.  They  appear  in  inflammations,  particularly  stone  or  schistosome  infection. 
They  may  be  found  in  conditions  where  toxins  are  being  eliminated  through  the 
kidneys,  as  in  tuberculosis.  The  menstrual  period  of  women  must  be  kept  in  mind 
in  the  examination  of  urine  sediments. 

Epithelial  Cells. — For  morphology  of  cells  from  different  locations  see  illustration. 
It  is  almost  impossible  to  state  positively  the  origin  in  the  genito-urinary  tract  of 
certain  cells.  Very  trustworthy  evidence  however  is  finding  of  epithelial  cells  on 
casts  or  the  so-called  compound  granine  cells  (fatty  degenerated  renal  epithelium). 
Sheets  of  more  or  less  small  round  or  caudate  epithelial  cells  are  rather  significant  of 
pyelitis.  Vaginal  epithelium  resembles  that  gotten  from  scraping  the  buccal 
mucosa. 

Of  casts  we  have  (i)  hyaline,  narrow  and  homogenous.  They  do  not  prove 
nephritis.  (2)  Epithelial  casts.  Usually  indicative  of  nephritis  but  very  slight 
inflammatory  processes  can  cause  them.  (3)  Blood  casts.  (4)  Granular  casts. 
If  coarse  granules  rather  significant  of  chronic  nephritis.  Finely  granular  casts  do 
not  seem  to  have  any  more  significance  than  hyaline  ones.  As  a  matter  of  fact 
under  dark  ground  illumination  hyaline  casts  show  a  granular  structure.  (5)  Waxy 
casts  are  highly  refractile,  show  fissuring  of  margins  and  are  of  serious  prognostic 
import  (chronic  nephiitis). 

Cylindroids  are  drawn  out  bodies  showing  tapering  ends,  irregularity  of  diameter 
and  longitudinal  striations. 

It  will  be  found  that  a  2/3  in.  objective  gives  almost  all  the  information 
required  as  to  casts.  It  is  quicker  and  gives  more  positive  information. 

Mounting  a  sediment  in  Gram's  solution  or  tinging  it  with  the  merest  trace  of 
neutral  red  is  of  much  assistance. 


344 


THE    URINE 


Special  features 

Sudden  onset.  CEdema 
often  marked,  especially 
of  face.  Mild  or  even 
severe  uraemic  symptoms. 
Pulse  tension  increased 
but  heart  not  hyper- 
trophied. 

Marked  oedema.  Uraemia 
common.  Hypertrophied 
left  ventricle.  Blood  pres- 
sure increased. 

No  oedema  until  later. 
High  blood  pressure  (200 
to  250).  Cardiac  hyper- 
trophy; often  uraemia  and 
albuminuric  retinitis. 

No  uraemia.  Symptoms 
attributable  to  heart. 

Reaction  of  urine  acid. 
No  tenesmus. 

Reaction  of  urine  alkaline 
or  very  faintly  acid. 
Tenesmus. 

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CHAPTER  XXVIII. 
THE  FJECES. 

IT  is  advisable  to  examine  a  stool  macroscopically  befoie  taking  up 
the  miscroscopical  examination.  The  mucus  shreds  or  casts  of  the 
bowel  in  mucous  colitis  or  membranous  enteritis  may  give  the 
diagnosis  of  obscure  abdominal  pain.  Pus  in  stools  may  often  be  noted 
without  the  aid  of  the  miscroscope. 

The  normal  stool  is  sausage  shaped  and  soft.  Neither  the  special  form  of  scybal- 
ous  masses  called  sheep  pellets  nor  the  pencil-like  nor  the  tape-like  excrement  prove 
the  existence  of  stricture  of  the  intestinal  lumen  although  suggestive  of  such  a  con- 
dition. The  mucus  of  bacillary  dysentery  is  opaque  and  grayish  from  the  great 
number  of  pus  and  phagocytic  cells.  It  is  well  to  remember  that  Charcot  Leyden 
crystals,  which  are  practically  always  absent  from  bacillary  dysentery  stools,  are 
not  infrequent  findings  in  the  amoebae  containing  stools;  of  course,  these  crystals 
appear  in  other  intestinal  parasite  infections. 

In  obstruction  of  the  common  bile  duct  we  have  acholic,  whitish,  foul-smelling 
stools.  If  the  putty  color  be  due  to  bacterial  change  exposure  to  the  air  will  restore 
the  brownish  tinge. 

Sprue  stools  are  white-wash  to  putty  colored,  pultaceous,  and  filled  with  air 
bubbles.  The  amount  is  excessive. 

Fatty  stools  are  best  examined  microscopically. 

As  so  many  solid  masses  resemble  gall-stones  it  is  well  to  dissolve  the  suspected 
mass  in  hot  alcohol  and  examine  for  cholesterin  crystals  upon  evaporation  of  the 
alcohol. 

If  the  faecal  examination  is  to  be  made  for  the  diagnosis  of  amoebae, 
in  a  case  where  the  characteristic  mucus  stools  are  not  present,  or  to 
verify  the  existence  of  flagellates,  it  is  best  to  give  a  dose  of  salts  early 
in  the  morning  and  examine  the  liquid  stools  which  follow  such  treat- 
ment. This  treatment  is  satisfactory  for  examination  for  intestinal 
parasites  or  ova. 

A  very  practical  way  of  obtaining  amoebae  is  to  pass  a  rectal  tube  or  a  piece  of 
drainage  tube  with  fenestrations  into  the  bowel,  and  amoebae  may  be  found  in  the 
mucus  filling  the  perforations  in  the  tube. 

If  the  purpose  of  the  examination  is  to  determine  the  digestive  power 
of  the  alimentary  tract  for  proteids,  carbohydrates,  or  fats,  it  is  best 
to  use  a  test  diet,  as  that  of  Schmidt  and  Strasburger. 

345 


346 


THE    FAECES 


Prior  to  using  this  test  diet,  one  should  familiarize  himself  with  the  macroscopic 
and  microscopic  appearances  resulting  from  such  a  diet  in  a  normal  person;  informa- 
tion is  then  at  hand  to  judge  of  variations  from  the  normal.  The  examination  of 
the  faeces  of  persons,  on  ordinary  and  specifically  undetermined  articles  of  diet,  is 
very  unsatisfactory  when  the  state  of  digestion  of  muscle  fibers  and  the  question 
of  fat  digestion  are  at  issue. 

In  examining  the  faeces  of  the  normal  person  and  likewise  with  the  patient,  wait 
until  the  second  or  third  day  so  that  the  faeces  of  previous  diets  may  have  passed  out. 
A  charcoal  powder  taken  before  commencing  the  diet  serves  as  an  indicator. 

Diet:  breakfast,  7  A.  M.,  bowl  of  oatmeal  gruel  (40  grams  oatmeal,  10  grams 
butter,  200  c.c.  milk,  300  c.c.  water).  Also  one  very  soft-boiled  egg  (i  min.)  and  50 
grams  zwieback.  In  the  forenoon,  500  c.c.  of  milk. 

For  dinner,  2  o'clock,  chopped  beef  broiled  very  rare  (125  grams  with  20  grams 
butter  poured  over  it).  Also  a  potato  puree  (200  grams  mashed  potato,  50  grams 
milk,  10  grams  butter).  Also  1/2  liter  of  milk  and  50  grams  zwieback. 


FIG.  104. — Microscopical  constituents  of  faeces,  (v.  Jaksch.}  a,  Muscle  fibers; 
b,  connective  tissue;  c,  epithelium;  d,  leukocytes;  e,  spiral  cells;  /,  g,  k,  i,  various 
vegetable  cells;  k,  "triple  phosphate"  crystals;  /,  woody  vegetable  cells;  the  whole 
interspersed  with  innumerable  microorganisms  of  various  kinds. 

For  supper,  7  o'clock,  the  same  articles  as  for  breakfast. 

This  detailed  diet  may  be  varied  to  suit  circumstances  as  regards  interchanging 
meals.  Furthermore,  the  milk  may  be  taken  in  the  form  of  tea  or  cocoa  or  cooked 
with  the  other  food.  Even  a  small  amount  of  wine  may  be  permitted.  The  diet 
taken,  however,  should  absolutely  conform  to  the  following  requirements:  i.  the 
taking  of  1/4  pound  chopped  beef,  a  portion  of  which  should  be  half  raw;  2.  the  milk 
taken  should  amount  to  about  a  quart;  3.  about  4  ounces  of  bread  or  toast  and  from 
4  to  8  ounces  of  potato  puree  should  be  eaten  daily. 

The  detailed  diet  contains  about  no  grams  albumin,  105  grams  fat  and  200 
grams  carbohydrates  with  a  fuel  value  of  2247  calories. 

The  stool  is  best  collected  in  quart  fruit  jars  and  examined  as  soon  after  evacua- 
tion as  possible.  The  wooden  spatula  like  tongue  depressors  are  well  adapted  to 
handling  the  specimen. 


FAT   IN   THE   F^CES  347 

Having  familiarized  one's  self  with  the  degree  of  digestion  of  muscle,  starch, 
and  fat  in  a  normal  person,  we  are  in  a  position  to  judge  of  the  state  of  assimilation 
in  a  patient. 

The  first  part  of  the  test  is  the  macroscopical  one.  For  this  grind  up  a  faecal 
mass  of  1/2  to  i  inch  diameter  in  a  mortar,  gradually  adding  water  until  it  has  the 
consistence  of  a  broth.  About  1/2  c.c.  of  this  emulsion  should  now  be  squeezed 
out  between  two  slides  and  studied  against  a  dark  surface  and  then  when  held  up 
to  the  light.  The  normal  stool  gives  a  rather  uniform  brownish  homogeneous  layer. 
Connective-tissue  remnants  (indicative  of  gastric  derangement)  show  as  whitish 
fibers.  Undigested  muscle  tissue  remnants  as  reddish-brown  splotches.  Fat 
particles  as  whitish-yellow  clumps.  Potato  remnants  appear  like  sago  grains  and 
mash  out  easily  like  mucus.  Mucus  is  best  noted  in  the  fecal  mass  before  making 
the  emulsion.  In  the  microscopical  test  of  this  emulsion. 

We  judge  of  muscle  digestion  by  the  intactness  of  the  striations. 
If  a  muscle  remnant  is  only  a  homogeneous  yellowish  particle,  it  shows 
satisfactory  digestion.  If  it  is  rectangular,  with  well-defined  cross 
striations,  it  shows  poor  digestion  for  meat  (Azotorrhcea).  A  loopful 
of  faeces  should  be  smeared  into  a  drop  of  Gram's  solution  for  starch- 
digestion  determination.  Normally  there  should  be  no  blue-staining 
starch  granules. 

Soaps  are  gnarled  bodies  everted  like  the  pinna  of  an  ear,  while  soap  crystals 
are  comparatively  coarse  and  do  not  melt  on  application  of  gentle  heat  as  do  the  more 
delicate  fatty  acid  crystals.  Neutral  fat  is  in  round  or  irregular  globules.  The  best 
stain  for  fat  is  Sudan  III  (saturated  solution  of  Sudan  III  in  equal  parts  of  70% 
alcohol  and  acetone). 

Mix  up  the  fasces  with  dilute  alcohol  (50  to  70%)  and  then  add  a  drop  of  the  above 
solution  and  apply  a  cover-glass  quickly.  The  fat  globules  show  as  orange  or 
golden-yellow  bodies. 

By  rubbing  up  a  small  portion  of  the  faeces  in  36%  acetic  acid,  applying  a  cover- 
glass  and  heating  over  a  flame  until  the  preparation  shows  bubbles,  we  convert  the 
soaps  and  other  fat  combinations  into  free  fatty  acids  which  show  as  more  or  less 
numerous  highly  refractile  bodies  which  show  a  crystalline  structure  as  the  prepara- 
tion cools.  By  practice  one  learns  the  amount  of  such  globules  to  expect  with  differ- 
ent fat  contents  in  stools. 

Steatorrhoea,  or  the  presence  of  fat  in  abnormal  quantities  in  the 
faeces,  is  shown  by  the  pale,  bulky,  greasy  stools  as  well  as  in  the  micro- 
scropical  examination. 

Average  for  normals  in  i  gm.  dried  fasces: 


Total  fat, 
Total  fatty  acid, 
Total  soap, 
Total  neutral  fat, 

225  mg. 
86  mg. 

74-7  mg- 
64.4  mg. 

(22.5%) 
(37.  9%  of  all  fat) 
(33.  4%  of  all  fat) 
(28.  5%  of  all  fat) 

348  THE    FAECES 

In  normal  cases  the  only  fat  elements  recognizable  are  yellow  calcium  or  color- 
less soaps. 

As  quantity  of  fat  increases  (as  say  500  to  600  mg.)  droplets  of  neutral  fat  appear 
with  or  without  increase  in  number  of  soap  masses.  Also  needles  and  splinters 
of  fatty  acid  and  soaps  appear.  Much  connective-tissue  debris  shows  defect  in 
gastric  digestion,  as  only  the  stomach  digests  connective  tissue. 

A  test  for  activity  of  fermentation  should  be  made  by  using  a  Schmidt 
apparatus. 

A  distinct  evolution  of  gas  in  twelve  hours  shows  starch  digestion  defect.  Such 
fasces  are  acid.  A  delayed  production  of  gas  (after  twenty-four  hours)  shows  albu- 
min decomposition.  Such  faeces  show  an  alkaline  reaction.  The  apparatus  is 
shown  in  Fig.  7.  Into  a  stocky  salt  mouth  bottle  we  put  approximately  5  grams 
of  faeces  which  have  been  rubbed  up  into  an  emulsion  with  water  and  fill  the  bottle 
with  water.  The  remaining  portion  of  the  apparatus  consists  of  a  test-tube  or  a 
graduated  cylinder  fitted  with  a  doubly  perforated  rubber  stopper.  One  U-shaped 
glass  tube  passing  through  this  stopper  connects  with  a  second  test-tube.  This  tube 
serves  as  a  receptacle  for  any  water  which  may  come  over  from  the  water-filled  tube 
or  graduated  cylinder  and  has  an  opening  punched  out  of  the  bottom  of  the  test- 
tube.  The  other  opening  in  the  twice  perforated  cork  admits  a  straight  tube  which 
connects  with  a  large  rubber  stopper  which  fits  into  the  bottle  for  the  fasces.  To 
prepare,  fill  the  graduated  cylinder,  then  push  in  the  doubly  perforated  cork  which 
is  connected  with  the  side  receiving  tube  and  the  large  rubber  cork.  This  latter  is 
then  pushed  down  to  fit  tightly  into  the  bottle  filled  full  with  the  faeces  emulsion. 

In  addition  to  the  faeces  examination  we  should  check  the  results  from  the  test 
diet  with  indican  and  nitrogen  partition  determinations  of  the  twenty-four  hour 
urine  specimen — the  ratio  of  ammonia  nitrogen  to  total  nitrogen  indicating  the  func- 
tional power  of  liver  and  the  indican  the  question  of  stasis  in  lower  part  of  small 
intestine. 

The  most  satisfactory  test  for  bile  in  the  faeces  is  to  emulsify  a  small 
particle  of  faeces  in  a  saturated  aqueous  solution  of  bichloride  of  mercury, 
preferably  with  a  wooden  tooth  pick,  on  a  concave  glass  slide.  After 
one  or  more  hours  hydrobilirubin-containing  faeces  show  a  salmon  pink 
color  and  bilirubin  ones  a  green  color.  One  should  familiarize  himself 
with  these  reactions  in  normal  cases. 

In  examining  a  liquid  stool  after  salts,  it  is  well  to  color  the  drop  of  faeces,  which 
is  to  be  covered  with  the  cover-glass,  with  a  small  loopful  of  1/2%  solution  of  neutral 
red.  If  diluting  fluid  is  used,  it  should  be  salt  solution,  and  not  water.  The  neutral 
red  tinges  the  granules  of  the  endoplasm  of  amoebae  and  flagellates  a  very  striking 
rose  pink  color,  thus  differentiating  them  from  vegetable  cells  or  body  cells. 

Whether  examining  the  thin  faeces  or  the  mucus  particle,  it  is  well  to  reserve 
report  on  amoebae  or  flagellates  until  motion  is  observed.  Encysted  protozoa  are 
difficult  to  diagnose. 

When  a  smear  preparation  is  desired,  we  may  smear  out  a  fragment  of  mucus 
and  stain  by  Romanowsky's  or  Gram's  method.  The  character  of  the  bacteria 


BACTERIAL  EXAMINATION   OF   FAECES  349 

present  appears  to  be  of  diagnostic  value — especially  in  the  case  of  infants  and  young 
children.  Beautiful  preparations  may  be  made  by  mixing  the  faeces  with  water,  then 
centrif uging  for  one  minute.  This  throws  down  vegetable  debris  and  crystals.  Now 
decant  the  supernatant  fluid,  which  holds  the  bacteria  in  suspension,  and  add  an 
equal  amount  of  alcohol.  Again  centrifuge,  decant,  and  smear  out  and  examine 
the  bacterial  sediment. 

Simply  taking  a  small  mass  of  faeces  and  emulsifying  it  with  a 
wooden  toothpick  on  a  concave  slide  in  70%  alcohol — then,  after  the 
sediment  settles,  taking  up  a  loopful  with  platinum  loop  from  the  sur- 
face and  smearing  out,  gives  a  very  satisfactory  smear.  Gram's  method, 
with  dilute  carbol  fuchsin  counterstaining,  gives  the  best  picture. 

The  Boas-Oppler  bacillus  may  be  found  in  the  stools  in  this  way.  Normally, 
a  Gram-stained  stool  shows  a  great  preponderance  of  Gram  negative  bacilli  and  such 
a  finding  in  a  measure  excludes  cancer  of  the  stomach.  Organisms  which  are  Gram 
positive  as  well  as  the  Boas-Oppler  bacillus  are,  i.  Lactic  acid  bacilli — these  show 
Gram  negative  areas  in  the  slender  bacilli.  2.  A  type  of  bacillus  similar  in  size  to  the 
colon  bacillus  but  Gram  positive  and  noncultivable  (found  in  acid  stools). 
3.  Bacilli  of  the  B.  subtilis  type. 

It  is  very  important  to  examine  the  fasces  for  T.  B.  With  children  a  diagnosis 
of  tuberculosis  may  be  made  in  this  way  when  the  sputum  cannot  be  obtained, 
the  pulmonary  secretion  being  swallowed.  The  preparation  on  the  concave  slide 
as  described  above  should  be  stained  for  T.  B. 

To  culture  for  typhoid,  dysentery,  cholera,  or  other  bacteria,  take  up  the  material 
in  a  tube  of  sterile  bouillon  and  smear  it  out  with  a  swab  over  a  lactose  litmus  agar 
plate  or  an  Endo  or  Conradi-Drigalski  plate.  Before  streaking  the  plates  they  should 
be  very  dry  on  the  surface.  This  can  be  best  done  by  pouring  into  a  plate  with  a 
circular  piece  of  filter-paper  in  the  lid  and  placing  in  the  incubator  for  one-half  hour 
to  dry.  The  filter-paper  absorbs  the  moisture.  Then  inoculate  the  surface  of  the 
plate  with  the  faecal  material. 

In  summer  complaints  of  infants  and  children  the  organisms  con- 
cerned are  as  a  rule  related  to  various  dysentery  strains  of  bacilli. 
Kendall  in  293  stool  examinations  found  the  gas  bacillus  (B.  aerog^ 
capsul.)  in  22  cases.  The  gas  bacillus  produces  intestinal  disorders 
which  are  not  benefited  by  lactose  but  by  buttermilk  (lactic  acid  bac- 
teria). For  diagnosis,  a  loopful  of  the  faeces  is  emulsified  in  a  tube  of 
sterile  milk  or  litmus  milk.  The  emulsion  is  heated  to  80°  C.  and  held  at 
this  temperature  for  20  minutes.  After  incubation  for  1 8  to  24  hours, 
preferably  anaerobically,  we  get  (i)  a  shreddy  disruption  of  the  casein, 
(2)  the  smell  of  rancid  butter  and  (3)  fully  80%  of  the  casein  is  dissolved. 
Smears  show  short  thick  Gram  positive  rods  with  slightly  rounded  ends. 
B.  Subtilis  is  sometimes  found  but  does  not  give  a  rancid  odor  nor  the 
strong  disruption  of  the  clot. 


350  THE  FAECES 

It  was  until  recently  thought  that  Cammidge's  reaction  (urine)  when  associated 
with  azotorrhoea  and  steatorrhcea  made  for  a  diagnosis  of  chronic  pancreatitis. 
At  present  very  little  importance  is  attached  to  the  Cammidge  reaction. 

Loss  of  weight,  anaemia,  diarrhoea  and  pains  in  the  upper  abdomen  are  important 
indications  of  pancreatic  trouble.  As  chronic  pancreatitis  is  often  associated  with 
cholelithiasis  jaundice  is  frequently  present.  Glycosuria  is  not  often  present. 
While  functional  tests  are  important  they  do  not  make  for  a  sure  diagnosis. 

Miiller's  method  for  pancreatic  functioning  determination  is  to  give  a  calomel 
purge  two  hours  after  a  meal.  A  little  of  the  liquid  stool  is  smeared  on  the  surface 
of  blood-serum  and  the  tube  incubated  at  60°  C.  (paraffin  oven).  If  the  surface  is 
smooth,  no  trypsin  was  present;  if  dotted  with  spots  of  digestion  liquefaction,  it 
shows  that  the  pancreatic  secretion  is  present. 

In  Schmidt's  nucleus  test  small  cubes  of  beef  are  hardened  in  absolute  alcohol 
and  then  tied  up  in  tiny  silk  bags.  These  are  recovered  from  the  faeces  and  sec- 
tioned. Complete  preservation  of  nuclei  indicates  a  total  absence  of  pancreatic 
functioning  provided  the  passage  of  the  tissue  be  not  too  rapid  as  by  diarrhoea. 

Epithelial  cells  are  generally  more  or  less  disintegrated.  In  the  mucus  of 
•bacillary  dysenteric  stools,  however,  large  intact  phagocytic  cells  are  frequent, 
which  may  be  mistaken  for  encysted  amoebae. 

Triple  phosphate  crystals  are  frequently  observed  in  faeces,  as  may  also  be  crys- 
tals of  various  calcium  salts.  Charcot-Leyden  crystals  are  rather  indicative  of 
helminthiases. 

Various  flagellates,  and  in  particular  Lamblia,  may  be  responsible  for  diarrhceal 
conditions  which  may  cause  rather  serious  symptoms. 

Balantidium  coli  has  been  reported  several  times  as  the  cause  of  dysenteric 
conditions.  Coccidiadea  are  found  in  the  fasces. 

It  is  in  the  faeces  we  examine  either  for  the  parasites  or  for  their  ova 
in  connection  with  practically  all  the  flukes,  except  the  lung  fluke  and 
the  bladder  fluke;  for  intestinal  taeniases  and  for  practically  all  the 
round  worms,  except  the  filarial  ones. 

Bass  has  recommended  that  faeces  which  have  been  made  fluid  be  centrifuged 
and  the  supernatant  fluid  containing  vegetable  debris  be  poured  off.  The  sediment 
contains  hookworm  eggs.  Then  pour  on  sediment  a  calcium  chloride  solution  of 
sp.  gr.  1050.  Again  centrifuge  and  decant.  Next  add  calcium  chloride  solution 
of  a  sp.  gr.  of  1250  and  centrifuge.  This  brings  to  the  surface  the  hookworm  eggs 
which  may  be  pipetted  off.  As  a  rule,  the  finding  of  hookworm  eggs  is  very  easy 
without  such  a  technic. 

In  the  tropics,  the  examination  of  the  faeces  vastly  exceeds  in  value 
that  of  urine  and  is  possibly  more  important  than  blood  examinations. 

The  larvae  of  various  insects  may  at  times  be  detected  in  the  stools,  as  well  as 
certain  acarines  (cheese  mites,  etc.). 

The  test  for  occult  blood  is  indicated  in  helminthiases  as  well  as  in  the  conditions 
for  which  it  is  usually  tested. 


CHAPTER  XXIX. 
BLOOD  CULTURES  AND  BLOOD  PARASITES. 

CLINICALLY,  the  most  important  examinations  of  the  blood  for 
parasites  is  for  the  presence  of  various  bacterial  infections  and  for 
certain  blood  protozoa  and  also  filarial  embryos. 

The  modern  method  of  culturing  blood,  especially  for  the  detection  of  typhoid 
or  paratyphoid  bacilli,  is  by  the  use  of  the  bile  media  of  Conradi.  Test-tubes  are 
filled  with  7  to  10  c.c.  of  i%  peptone  ox  bile,  or  ox  bile  alone,  and  the  medium  is 
sterilized  in  the  autoclave.  It  is  good  practice  to  place  the  syringe  in  a  plugged 
test-tube  containing  salt  solution,  with  the  needle  unscrewed.  After  autoclaving, 
the  sterile  syringe  can  be  taken  to  the  bedside  in  the  test-tube.  Using  a  wide  test- 
tube,  a  forceps  can  be  sterilized  at  the  same  time  and  used  to  attach  the  needle  to  the 
barrel  of  the  syringe. 

By  using  a  piece  of  glass  tubing  into  which  the  needle  is  inserted  we  may  sterilize 
the  syringe  easily  in  the  test-tube.  The  glass  tubing  prevents  the  steel  needle  from 
coming  in  contact  with  the  glass  of  the  test-tube  and  so  prevents  cracking  the  test- 
tube.  Instead  of  a  syringe  a  better  apparatus  is  a  test-tube  or  an  Erlenmeyer  flask 
\yith  a  double  perforation  stopper  for  insertion  of  two  pieces  of  glass  tubing,  one 
joined  to  a  piece  of  rubber  tubing  carrying  the  needle  and  the  other  attached  to  a 
rubber  tube  for  suction  by  the  operator's  mouth.  See  Fig.  7. 

The  skin  should  be  scrubbed  gently  with  green-soap  solution  and  water  for  about 
three  minutes.  The  skin  of  the  area  to  be  punctured  should  then  be  sterilized  by 
the  gentle  application  of  Harrington's  solution  (not  scrubbed)  for  one-half  minute, 
and  should  then  be  washed  with  sterile  water.  It  appears  to  be  safe  to  simply 
scrub  the  area  with  70%  alcohol  for  one  or  two  minutes.  Applications  of  pure 
carbolic  acid  on  a  gauze  wad  for  a  few  seconds  followed  by  neutralization  with  70% 
alcohol  gives  satisfactory  sterilization.  The  present  method  of  sterilizing  skin 
for  taking  blood  or  inoculating  vaccines  is  simply  to  smear  the  site  of  entrance  for 
the  needle  rather  heavily  with  tincture  of  iodine.  A  tourniquet  is  now  applied  to 
distend  the  vein,  and  the  needle  is  inserted  in  the  direction  of  the  venous  flow. 
Withdrawing  5  to  10  c.c.  of  blood,  we  loosen  the  tourniquet,  (otherwise  the  blood 
may  flow  from  the  puncture)  then  withdraw  the  needle,  and  force  out  abo'ut  1/2  c.c. 
into  the  first  bile  tube,  about  i  c.c.  into  the  second,  and  2  or  3  c.c.  into  the  third. 
It  is  well  to  reserve  some  of  the  blood  for  Widal  tests. 

The  bile  tubes  are  now  incubated  for  ten  to  twelve  hours  and  then  transfers 
are  made  to  bouillon  tubes.  These  bouillon  tubes  can  be  used  in  six  to  eight  hours 
for  testing  the  organism  against  known  typhoid  or  paratyphoid  sera.  Test-tubes 
containing  10  c.c.  of  ordinary  bouillon  with  i%  of  sodium  citrate  are  as  satisfactory 
as  bile  media. 

351 


352         BLOOD  CULTURES  AND  BLOOD  PARASITES 

Some  prefer  a  2%  sodium  glycocholate  in  bouillon  while  others  use  a  2%  solution 
of  ammonium  oxalate  in  bouillon  for  blood  cultures. 

Some  prefer  to  streak  plates  of  lactose  litmus  agar  with  material  from  the  bile 
tubes  instead  of  inoculating  the  bouillon  tubes.  Contamination  with  staphylococci 
or  the  presence  of  staphylococci,  streptococci,  or  plague  bacilli  in  septicagrmc  con- 
ditions show  easily  accessible  colonies. 

Schotmuller  adds  i  to  3  c.c.  of  blood  to  liquefied  agar  at  45°  C.,  and  after  mixing 
pours  into  plates.  The  standard  method  formerly  was  to  add  the  blood  to  an 
excess  of  bouillon  (i  to  5  c.c.  of  blood  to  100  c.c.  or  more  of  bouillon). 

A  very  useful  procedure  in  the  isolation  of  streptococci,  pneu- 
mococci,  plague  and  anthrax  bacilli  is  to  inject  i  to  2  c.c.  of  blood 
into  suitable  animals.  When  injecting  mice  use  only  about  0.2  c.c. 

By  using  the  bile  media,  we  can  take  the  blood  from  the  ear  in  typhoid  cases, 
if  preferred.  Then  if  chance  staphylococcic  contamination  occurs,  such  colonies 
are  readily  differentiated  from  typhoid  ones  by  the  pink  color  on  lactose  litmus 
agar.  For  culturing  blood  in  septicaemic  conditions,  the  blood  should  always  be 
drawn  from  the  vein  and  cultured  either  by  mixing  i  to  2  c.c.  with  melted  agar 
and  then  pouring  plates  or  by  transferring  to  bouillon  in  excess  (at  least  ten  times 
as  much  bouillon  as  blood)  and  after  eighteen  to  twenty-four  hours'  incubation 
plating  out.  For  streptococcus  and  pneumococcus  blood  agar  plates  are  to  be  pre- 
ferred, the  pneumococcus  giving  green  colonies  with  only  a  suggestion  of  haemolysis 
while  the  streptococcus  gives  an  opaque  colony  with  a  distinct  haemolytic  zone 
surrounding  it. 

Typhoid  cultures  are  best  obtained  in  the  first  week  of  the  disease, 
after  that  time  the  Widal  is  the  test  of  preference. 

If  a  paratyphoid  serum  is  not  at  hand  for  testing,  it  may  suffice  to  inoculate  a 
glucose  bouillon  tube  or  a  Russell  lactose  glucose  litmus  slant;  gas  production 
indicates  paratyphoid.  This  test  should  be  applied  when  a  very  motile  organism 
does  not  show  agglutination  with  a  known  typhoid  serum.  Anthrax  and  glanders 
should  be  considered  in  blood  cultures. 

In  Malta  fever  it  must  be  remembered  that  colonies  do  not  show  themselves 
for  several  days.  Addition  of  blood  to  melted  agar  is  a  good  procedure. 

Blood  for  culturing  typhoid  or  the  paratyphoids  may  be  taken 
with  a  Wright's  tube  from  the  ear  or  finger.  Dipping  the  hand  in  hot 
water  assists  the  flow  of  blood.  The  supernatant  serum  after  centrifu- 
galization  should  be  pipetted  off  with  a  sterile  pipette  and  reserved 
for  agglutination  tests  while  the  clot  is  dropped  into  a  bile  tube. 
(Clot  culture.) 

Rosenberger  was  the  first  to  insist  upon  the  importance  of  examination  of  blood 
for  T.  B.  Brem  considered  that  many  cases  of  finding  of  acid-fast  bacilli  were  not 
of  T.  B.  The  Kurashigi-Schnitter  method  for  tubercle  bacilli  in  blood  is  to  take 


BLOOD  PARASITES  353 

about  i  c.c.  blood  and  put  in  a  centrifuge  tube  containing  5  c.c.  of  3%  acetic  acid. 
After  the  red  cells  are  thoroughly  laked  centrifuge,  pipette  off  supernatant  fluid  and 
dissolve  the  sediment  in  5  c.c.  antiformin.  When  dissolved  add  5  c.c.  absolute 
alcohol  and  centrifugalize  for  twenty  minutes.  Smear  out  the  sediment  and  stain. 

The  examination  of  the  blood  for  the  parasites  of  malaria,  filariases,  kala-azar 
and  spirillum  fevers  has  been  discussed  under  their  respective  headings. 

With  trypanosomes  from  human  trypanosomiasis,  smears  from  gland  juice  or 
cerebrospinal  fluid  seem  more  satisfactory  to  examine  than  blood  smears  unless  the 
blood  is  taken  in  5  to  10  c.c.  quantities  and  centrifuged  in  sodium  citrate  salt  solution. 

The  latest  method  in  the  diagnosis  of  trichinosis  is  to  take  5  to  10  c.c.  of  blood 
from  a  vein  at  the  time  of  the  migration  of  the  embryos  to  the  muscles  (10  to  20 
days).  This  is  forced  out  into  a  centrifuge  tube  containing  3%  acetic  acid,  and 
the  sediment  examined  for  trichina  larvae. 


CHAPTER  XXX. 
THE  STOMACH  CONTENTS. 

FROM  a  microscopical  standpoint  there  is  comparatively  little  that 
is  of  value  in  the  examination  of  the  gastric  contents;  there  is  nothing 
very  specific  about  the  findings. 

A  test  meal  is  not  a  necessity  as  in  the  chemical  examination,  but 
either  vomitus  or  material  withdrawn  with  a  stomach-tube  two  or  more 
hours  after  an  ordinary  meal  suffice. 

The  most  satisfactory  specimen  is  one  taken  before  the  giving  of 
the  test  meal. 

The  washings  from  the  stomach  are  allowed  to  stand  until  the 
sediment  has  fallen  to  the  bottom  and  an  examination  of  this  is  made. 

The  microscopical  diagnostic  points  in  connection  with  distinguishing  cancer  of 
the  stomach  from  nonmalignant  dilatation  are:  i.  Fragments  of  cancer  tissue. 
These  are  very  rarely  found  and  are  most  difficult  to  diagnose.  2.  The  presence  of 
flagellates  in  the  early  stages  of  cancer  (the  so-called  anacid  stage  preceding  the 
development  of  lactic  acid) .  As  flagellates  prefer  an  alkaline  medium,  they  disappear 
after  the  acidity  due  to  lactic  acid  comes  on.  3.  The  presence  of  the  Boas-Oppler 
bacillus.  There*  are  probably  several  organisms  so  designated.  They  are  lactic  acid 
producers  and  are  characterized  by  being  very  large  bacilli  (7X1/0  and  arranged  in 
long  chains  which  stretch  across  the  field  of  the  microscope.  They  are  Gram  posi- 
tive and  do  not  form  spores.  They  can  be  cultivated  on  media  rich  in  blood  and 
are  aerobic.  They  should  only  be  reported  when  present  in  great  abundance  and  in 
long  chains.  Heinemann  thinks  it  probable  that  the  Boas-Oppler  bacillus,  Lepto- 
thrix  buccalis,  and  B.  bifidus  may  be  identical  with  B.  bulgaricus  (see  under  Milk). 
4.  The  absence  of  sarcinae  and  yeasts.  The  presence  of  these  sarcinae  and  fungi  in 
vomitus  is  indicative  of  a  simple  dilatation. 

In  chronic  gastritis  the  picture  of  mucus  entangling  large  numbers  of  epithelial 
cells  is  characteristic. 

In  examining  the  sediment  from  the  filter-paper  after  filtering  off  the  stomach 
contents  always  use  a  dilute  Gram  solution  (about  i  to  4)  for  mounting  the  sediment. 
Muscle  fibers,  yeast  cells,  red  blood-cells,  and  epithelial  cells  are  stained  a  golden 
yellow.  Starch  granules  are  stained  blue  while  fats  are  unstained  and  show  as  glo- 
bules of  varying  sizes. 


354 


CHAPTER  XXXI. 
EXAMINATION  OF  PUS. 

Pus  may  be  collected  for  examination  either  i.  with  a  platinum  loop, 
2.  with  a  sterile  swab,  3.  with  a  bacteriological  pipette  or  4.  with  a 
hypodermic  syringe. 

It  is  always  well  to  make  a  smear  and  stain  it  by  Gram's  method  at 
the  same  time  that  cultures  are  made.  The  Gram  stain  gives  informa- 
tion as  to  the  abundance  of  organisms  in  the  pus  and  as  to  the  probable 
findings  in  the  culture.  Pneumococci  and  streptococci  are  differenti- 
ated from  the  staphylococci  in  this  way  without  the  necessity  of  more  or 
less  extended  cultural  methods. 

Smears  from  material  examined  for  gonococci  may  show  Gram  negative  diplo- 
cocci  which,  however,  do  not  generally  have  the  morphology  of  the  gonococcus. 
They  are  furthermore  extracellular. 

The  M.  catarrhalis  has  been  reported  from  urethral  smears  though  very  rarely. 
Diphtheroid  organisms  are  not  uncommon.  Gram  positive  cocci  are  rather  common 
in  smears  from  discharges  of  chronic  gonorrhoea. 

When  autogenous  vaccines  are  to  be  made,  the  isolation  of  the  exciting  organism 
is  necessary.  This  is  best  done  by  streaking  the  pus,  taken  up  with  a  sterile  swab 
and  emulsified  in  a  tube  of  bouillon,  over  the  surface  of  an  agar  plate.  Practically 
as  convenient  and  providing  a  more  nutritious  medium  is  to  smear  the  material  on 
a  loop  or  swab  over  the  surface  of  a  blood-serum  slant,  then  to  inoculate  a  second 
tube  from  the  first  without  recharging  the  loop  or  swab,  and  so  on  until  three  or  four 
tubes  are  inoculated.  Isolated  colonies  should  be  obtained  in  a  third  or  fourth  tube. 

In  examining  blood-serum  slants  inoculated  with  purulent  material,  always 
examine  the  water  of  condensation  for  streptococci. 

A  bacteriological  pipette  is  very  useful  when  pus  is  to  be  sent  to  a  laboratory; 
the  tip  can  be  sealed  in  a  flame  and  the  cotton  plug  at  the  other  end  insures  the 
noncontamination  of  the  contents.  The  material  may  be  drawn  up  either  with  the 
mouth  or  with  a  rubber  bulb. 

The  hypodermic  syringe  is  very  useful  in  puncturing  buboes,  etc., 
especially  in  plague.  A  small  pledget  of  cotton  on  a  toothpick  dipped 
into  pure  carbolic  acid  and  touched  to  a  spot  over  the  bubo,  which  after 
about  thirty  seconds  is  soaked  with  alcohol,  makes  a  sterile  anaesthetic 
spot  at  which  to  introduce  the  needle  of  the  syringe.  It  must  be  remem- 

355 


356  EXAMINATION   OF   PUS 

bered  that  when  plague  buboes  begin  to  soften,  the  plague  bacilli  may 
be  replaced  by  ordinary  pus  organisms. 

It  is  remarkable  how  frequently  we  get  pure  cultures  from  abscess  material.  In 
purulent  material  from  abdominal  abscesses  we  are  apt  to  obtain  mixed  cultures, 
especially  the  colon  bacillus  and  B.  pyocyaneus,  in  addition  to  ordinary  pus 
organisms. 

When  it  is  a  question  between  streptococci  and  pneumococci,  it  is  well  to  inocu- 
late a  mouse;  the  capsulated  pneumococci  at  the  autopsy  make  the  diagnosis. 

Animal  inoculation  is  also  necessary  in  plague  and  glanders,  and  possibly  anthrax. 
When  tetanus  is  suspected,  it  should  be  examined  for  as  described  under  Tetanus. 
Tuberculosis  should  also  be  identified  by  inoculating  a  guinea-pig,  as  well  as  by  acid- 
fast  staining  and  culture,  if  there  is  any  doubt  as  to  the  nature  of  the  material. 

The  black  or  yellow  granules  of  madura  foot,  as  well  as  those  of  actinomycosis, 
should  be  examined  as  recommended  in  the  section  on  fungi. 

Amcebae,  coccidia,  and  larval  echinococci  may  be  found  in  purulent  material, 
as  may  also  various  other  animal  parasites,  as  fly  larvae,  sarcopsyllae,  etc. 

The  pus  from  an  amoebic  abscess  of  the  livei  is  as  a  rule  sterile  when 
cultured. 

The  examination  at  the  time  of  operation  or  exploration  frequently 
shows  an  absence  of  amoebae  as  well  as  of  bacteria.  Two  or  three  days 
later  amoebae  may  be  found  in  the  pus  draining  from  the  abscess  cavity. 

Flukes,  round- worms,  and  whip- worms  may  as  a  result  of  their  wandering  from 
the  intestinal  lumen  cause  abscesses. 

Serious  ulcerations  may  follow  infection  with  the  Guinea-worm. 


CHAPTER  XXXII. 
SKIN  INFECTIONS. 

CULTURAL  methods  are  as  a  rule  to  be  preferred  in  the  bacterio- 
logical examination  of  the  skin. 

This  is  best  done  by  washing  the  surface  to  be  examined  with  soap  and  water,  in 
order  to  eliminate  chance  organisms  which  may  have  settled  on  the  surface  of  the 
skin  in  dust  or  as  a  result  of  contact  with  material  containing  them.  Scrapings  are 
then  made  with  a  sterile  dull  scalpel,  and  this  material  is  emulsified  in  a  drop  of 
sterile  water  in  the  center  of  a  Petri  dish.  A  tube  of  melted  agar  at  42°  C.  is  then 
poured  on  the  inoculated  drop  and,  by  mixing,  the  bacterial  flora  is  distributed  over 
the  entire  surface  of  the  plate.  Of  the  colonies  developing  on  such  plates  probably 
80%  will  be  found  to  be  staphylococci,  and  of  these  the  greater  proportion  will 
be  staphylococci  showing  white  colonies. 

Occasionally  the  aureus  or  citreus  may  be  isolated. 

Streptococci  and  colon  bacilli  are  rarely  found. 

The  Staphylococcus  pyogenes  aureus  is  the  organism  usually  isolated  from 
furuncles,  circumscribed  abscesses,  and  carbuncles. 

Streptococci  are  the  organisms  to  be  expected  in  phlegmonous  infections. 

Cold  abscesses,  which  are  frequently  due  to  tuberculous  infection,  are,  as  a  rule, 
sterile. 

Acne  pustules  may  show  staphylococci  or  the  microbacillus  of  acne  may  be 
present. 

The  Bacillus  acnes  is  a  short  broad  bacillus  often  showing  a  beaded 
appearance  when  stained  by  Gram's  method.  It  is  Gram  positive 
According  to  Hartwell  it  grows  readily  on  glucose  agar  when  cultivated 
anaerobically  (Wright's  method).  Colonies  appear  in  four  to  five  days. 

Sabouraud's  medium  for  its  culture  is:  Peptone  20  grams,  glycerine  20  grams, 
glacial  acetic  acid  5  drops,  agar  15  grams  and  water  1000  c.c.  The  bottle  bacillus, 
which  morphologically  resembles  a  yeast,  is  considered  to  be  the  cause  of  dry  pityri- 
asis  capitis.  It  may  also  be  found  in  the  comedones  of  children. 

In  the  tropics,  an  organism  which  at  times  produces  lesions  similar  to  impetigo 
and  again  pemphigoid  eruptions  and  at  other  times  wide-spreading  erysipelatous 
conditions  gives  cultural  characteristics  similar  to  S.  pyogenes  aureus.  It  is  probably 
only  a  virulent  aureus.  It  has  been  described  under  the  name  of  Diplococcus  pem- 
phigi  contagiosi. 

The  Staphylococcus  epidermidis  albus,  or  stitch  abscess  coccus,  is  considered  by 
Sabouraud  to  be  the  cause  of  eczema  seborrhoicum. 

357 


358  SKIN   INFECTIONS 

It  is  in  scrapings  from  the  skin  of  lepromata  that  we  find  acid-fast 
organisms  in  the  greatest  profusion.  In  tuberculosis  of  the  skin  the 
tubercle  bacilli  are  exceedingly  scarce.  Inoculation  of  a  guinea-pig 
will  piobably  give  positive  results  with  the  tubercle  bacillus.  The 
leprosy  bacillus  is  noninoculable  for  experimental  animals. 

Anthrax. and  glanders  cause  skin  lesions  which  can  only  be  surely  diagnosed 
culturally  or  by  animal  inoculation. 

Plague  bacilli  may  be  isolated  from  the  primary  vesicles  appearing  at  the  site  of 
the  flea  bite. 

Tropical  phagedaena  is  thought  by  some  to  be  due  to  a  sort  of  diphtheroid  or- 
ganism. The  organisms  of  Vincent's  angina  may  cause  tropical  ulcer. 

The  skin  diseases  due  to  fungi  are  discussed  under  that  section.  Of  the  skin 
affections  caused  by  animal  parasites,  ground  itch  is  the  most  important.  This  is  a 
form  of  dermatitis  due  to  the  irritation  set  up  by  the  hook-worm  larvae  penetrating 
the  skin  of  the  foot  and  leg. 

The  Sarcopsylla  penetrans  or  jigger  (sand  flea)  is  an  important  agent  in  ulcera- 
tions  about  the  foot. 

Certain  acarines  cause  skin  lesions,  as  is  also  the  case  with  the  larvae  of  certain 
flies. 

The  itch  mite  (Sarcoptes  scabiei)  is  an  important  animal  parasite  of  the  skin. 

The  various  lice,  fleas  and  bed  bugs  are  well  understood  as  causes  of  skin  irrita- 
tion. 

Filarial  infections  are  also  important  especially  the  ulcers  of  the 
Guinea-worm,  Calabar  swellings  of  F.  loa,  the  cystic  tumors  of  F. 
volvulus  and  the  varicose  groin  glands  and  elephantiasis  of  F.  bancrofti. 

Leeches,  as  H.  ceylonica,  may  cause  serious  ulceration. 

Oxyuris  may  cause  a  severe  irritation  about  the  region  of  the  groin  and  inner 
surfaces  of  the  thigh,  and  especially  about  the  vulvar  region  of  female  children. 

Gnathostomum  siamense,  a  nematode  with  two  lip-like  structures  and  spine- 
like  structures  covering  its  anterior  one-third,  has  been  found  once  in  a  tumefaction 
of  the  breast. 

Plerocercoid  larvae  of  Dibothriocephalidae  have  been  found  in  the  subcutaneous 
tissues. 

Certain  skin  diseases,  as  Oriental  sore  and  yaws,  are  protozoal  in  origin. 


CHAPTER  XXXIII. 
CYTODIAGNOSIS. 

THIS  method  of  diagnosis  is  chiefly  employed  in  the  examination  of 
cellular  sediments  of  pleural,  ascitic,  and  ceiebrospinal  fluid. 

The  fluids  which  pathologically  collect  in  the  serous  cavities  are 
divided  into  two  classes,  i.  the  transudates,  which  form  as  the  result 
of  some  circulatory  inadequacy  and  2.  the  exudates,  which  result  from 
inflammatory  processes. 

Transudates  have  little  or  no  fibrin  and  very  few  cellular  elements  and  do  not 
contain  nucleo-albumin.  Exudates  contain  nucleo-albumin  and  usually  have  a 
specific  gravity  above  1018,  while  that  of  the  transudates  is  lower  than  1018. 

There  are  two  simple  methods  for  differentiating  transudates  and  exudates. 
Moritz  adds  2  drops  of  a  5%  solution  of  acetic  acid  to  the  fluid  to  be  tested.  A 
heavy,  cloud-like  precipitate  shows  the  fluid  to  be  of  inflammatory  origin  (an 
exudate).  A  transudate  may  produce  a  slight  opalescence.  Rivalta's  test  consists 
in  dropping  a  drop  of  the  fluid  to  be  tested  into  a  cylinder  containing  2  drops  of 
glacial  acetic  acid  in  100  c.c.  distilled  water.  A  nebulous  cloud  as  the  drop  of  fluid 
sinks  shows  an  exudate. 

For  pleural  fluids  we  should  receive  the  material  in  centrifuge  tubes  about  one- 
fourth  filled  with  2%  sodium  citrate  salt  solution.  This  prevents  clotting.  Having 
thrown  down  the  sediment,  the  supernatant  fluid  is  poured  off,  and  in  its  place  a  i  % 
aqueous  solution  of  formalin  is  added.  After  mixing  and  allowing  to  stand  for  about 
five  minutes,  centrifugalization  is  again  repeated  and,  pouring  off  the  supernatant 
formalin  solution,  we  make  smears  from  the  sediment.  This  is  either  stained  by  a 
Romano wsky  method  or,  after  fixing  with  heat  (burning  alcohol),  the  smear  is 
stained  with  haematoxylin  and  eosin. 

With  ascitic  fluid  it  is  usually  sufficient  to  centrifuge  the  fluid,  then  decant  off  the 
supernatant  fluid  and  drain  by  means  of  a  piece  of  filter-paper  held  at  the 
mouth  of  the  upturned  tube.  The  sediment  adheres  to  the  bottom  of  the  tube  and 
is  best  emulsified  with  the  small  amount  of  fluid  remaining  by  means  of  a  bulb 
pipette.  The  material  is  sucked  up,  smeared  out  on  a  slide  with  a  second  slide  as 
for  blood  and  stained  preferably  with  Giemsa  after  fixation.  H.  E.  staining  brings 
out  mitotic  figures  best.  If  the  fluid  has  coagulated  it  is  best  to  take  a  little  of  the 
coagulum  and  stain  it  with  neutral  red  as  for  vital  staining.  It  is  difficult  to  disso- 
ciate the  cells  from  the  clot.  The  wet  Giemsa  method  described  for  blood  gives  good 
results  with  puncture  fluid  sediments. 

At  the  time  of  securing  fluid  for  cytodiagnosis,  cultures  should  be  made  on  blood- 

359 


360  CYTODIAGNOSIS 

serum  for  various  pyogenic  bacteria  and,  if  tuberculosis  is  suspected,  inoculation 
of  a  guinea-pig  is  indicated. 

The  interpretation  of  cellular  sediments  is  more  difficult  than  many  books  would 
indicate,  there  being  many  factors  which  tend  to  complicate  the  findings. 

The  polymorphonuclears  in  purulent  fluids  often  show  fatty  degeneration, 
swollen  and  faintly  staining  nucleus  or  a  breaking  up  of  the  nucleus  into  small  deeply 
staining  masses  (nuclear  fragmentation).  Such  fragments  in  the  smear  may  be  con- 
fusing. The  endothelial  cells  often  show  fatty  degeneration  in  the  cytoplasm  and 
we  often  note  bacteria  and  other  cells  which  have  been  phagocytized  by  them. 
Where  proliferation  of  endothelial  cells  is  going  on  actively  the  cells  show  a  rather 
deeply  staining  cytoplasm  as  compared  with  the  light  staining  cytoplasm  of  the  cells 
in  transudates. 

The  following  are  the  leading  differentiations: 

1.  A  smear  showing  almost  entirely  lymphocytes  with  a  few  red 
cells  and  very  rarely  a  polymorphonuclear  indicates  a  tuberculous 
process. 

2.  Where  a  pyogenic  process  is  engrafted  on  a  tuberculous  one,  we 
have  still  the  red  cells,  some  degenerated  lymphocytes,  and  in  particular 
polymorphonuclears  showing  fragmentation  of  their  nuclei. 

3.  When  a  hydro  thorax  results  from  chronic  heart  or  kidney  disease, 
the  characteristic  cell  is  the  endothelial  cell,  which  greatly  resembles  a 
large  mononuclear.     These  cells  often  are  arranged  in  plaques. 

4.  Some  authorities  consider  that  the  cancer  cell  can  be  recognized 
by  its  occurring  in  masses  and  having  a  markedly  vacuolated  cytoplasm. 
It  has  been  claimed  that  they  contain  glycogen  by  which  means  we  can 
distinguish  them  from  endothelial  cells  which  they  so  much  resemble. 
If  such  cells  should  show  mitosis  the  finding  would  be  suggestive. 
For  mitotic  figures  wet  fixation  with  some  bichloride  fixative,  with  H.  E. 
staining,  is  best. 

Jousset  introduced  inoscopy  as  a  means  of  diagnosing  tuberculosis.  The  fluid 
was  allowed  to  coagulate  and  was  then  digested  with  an  artificial  gastric  juice.  The 
digested  material  was  then  centrifuged  and  the  sediment  examined  for  tubercle 
bacilli.  This  process  does  not  seem  to  have  met  with  much  favor  in  this  country. 
(Using  sodium  citrate  obviates  the  necessity  for  digesting  the  coagulum.) 

The  same  points  will  hold  for  ascitic  fluid  as  for  pleural  fluid. 

In  taking  cerebrospinal  fluid  for  culture  and  cytodiagnosis  we  use  a  stout  anti- 
toxin needle  without  attaching  a  syringe.  Aspiration  is  responsible  for  many  of 
the  ill  effects  of  lumbar  puncture.  The  needle  should  be  about  4  inches  long  for  an 
adult.  Sterilize  the  skin  and  needle  as  described  for  blood  cultures  from  a  vein. 
To  make  a  lumbar  puncture,  place  patient  on  left  side  with  knees  drawn  up.  A  line 
at  the  level  of  the  iliac  crests  passes  between  the  third  and  fourth  lumbar  vertebrae. 
Select  a  point  midway  between  the  spinous  processes  of  these  lumbar  vertebrae  and 


CYTODIAGNOSIS   AND   PARASYPHILITIC   DISEASE  361 

enter  the  needle  2/5  of  an  inch  to  the  right  of  this  point,  pushing  the  needle  inward 
and  upward.  Collect  the  material  in  a  sterile  test-tube.  Make  cultures  on  blood- 
serum  and  then  centrifugalize  and  examine  the  sediment  as  for  pleural  fluids. 

In  general  terms  it  may  be  stated  that: 

1.  A  lymphocytosis  indicates  a  tuberculous  process. 

2.  An  abundance  of  polymorphonaclear  and  eosinophilic  leukocytes 
indicates  a  meningococcic,  streptococcic,  influenza  or  pneumococcic 
infection. 

When  the  case  is  one  of  meningism  there  are  very  few.  cells.  In  poliomyelitis 
there  is  a  cell  increase  of  which  90%  may  be  lymphocytes. 

A  method  of  examination  considered  by  neurologists  as  of  differential  diagnostic 
value  is  to  count  the  number  of  cells  in  a  cubic  millimeter  of  the  cerebrospinal  fluid. 
The  technic  is  to  use  a  gentian- violet- tinged  3%  solution  of  acetic  acid.  This  is 
drawn  up  to  the  mark  0.5,  and  the  cerebrospinal  fluid  is  then  sucked  up  to  n.  After 
mixing,  the  cell  count  is  made  with  the  haemocytometer.  Normally  we  have  only 
one  or  two  cells  per  cubic  millimeter,  but  in  tabes  or  general  paresis  this  is  increased 
to  50  or  100  cells  (greatest  at  onset  of  disease). 

The  test  for  globulins  as  showing  parasyphilitic  disease  is  taken  up  under  Tre- 
ponema  pallidum.  The  technic  of  the  Wassermann  test  with  cerebrospinal  fluid 
is  discussed  under  that  test.  Any  excess  of  urea  in  the  cerebrospinal  fluid  is  a  sure 
sign  of  renal  inadequacy. 

Trypanosomiasis  gives  a  cellular  increase  very  similar  to  syphilis. 

In  the  work  of  the  French  Sleeping  Sickness  Commission  five  cells  per  cubic 
millimeter  was  taken  as  normal. 


CHAPTER  XXXIV. 
RABIES,  VACCINIA  AND  THE  FILTERABLE  VIRUSES. 

RABIES  is  a  disease  of  dogs  and  wolves,  but  is  communicable  to  man 
and  domesticated  animals.  The  virus,  whatever  it  may  be,  resides  in 
the  saliva  and  nervous  structures.  It  is  destroyed  by  a  temperature  of 
50°  C.  In  man  the  period  of  incubation  is  usually  from  three  weeks  to 
three  months,  but  may  be  shorter  or  may  extend  over  one  year. 

Bites  about  the  face  and  those  with  marked  lacerations  are  particularly  serious. 
Bites  of  rabid  wolves  give  about  four  times  as  great  a  mortality  as  those  of  dogs. 
In  the  dog  there  are  two  types  of  the  disease — dumb  rabies  and  furious  rabies. 

By  inoculating  rabbits  subdurally  with  an  emulsion  of  the  brain  or  spinal  cord 
of  a  rabid  animal,  and  successively  the  medulla  of  this  rabbit  subdurally  into  other 
rabbits,  we  finally  so  increase  the  virulence  of  the  infection  that  rabbits  die  in  six  days. 
Beyond  this  it  is  impossible  to  increase  the  virulence  and  it  is  termed  "fixed  virus." 
The  pathogenic  power  of  this  virus  is  also  changed  so  that  it  is  not  apt  to  cause  rabies 
if  injected  subcutaneously.  To  attenuate  this  virus  the  spinal  cord  of  the  rabbit 
is  removed  and  is  dried  over  caustic  potash  at  a  temperature  of  23°  C.  The  cord  is 
divided  into  segments  about  i  inch  in  length.  Drying  for  about  fifteen  days  seems 
to  entirely  destroy  the  virus. 

To  prepare  the  material  for  prophylactic  injections  a  small  portion  of  the  cord  is 
emulsified  with  normal  salt  solution  and  injected  subcutaneously.  The  German 
method  is  to  commence  with  a  cord  that  has  been  desiccated  only  eight  days.  At 
first  injections  are  given  daily,  and  it  is  possible  to  inject  three  days'  cords  by  the 
sixth  day.  The  immunity  is  "active"  and  the  immunizing  agent  is  a  "vaccine." 
Like  vaccine  virus  the  product  can  be  preserved  (for  probably  a  month)  by  the  use 
of  glycerine  so  that  it  is  now  possible  to  send  the  material  for  inoculation  from  the 
laboratory  preparing  it. 

The  treatment  lasts  for  about  twenty  days.  In  the  diagnosis  of  rabies  in  dogs 
it  is  preferable  to  preserve  the  animal  so  that  the  development  of  the  symptoms 
may  be  observed. 

In  case  the  dog  has  been  killed,  it  may  be  possible  to  make  a  diagno- 
sis by  means  of  the  Negri  bodies.  These  are  round  or  oval  bodies  from 
i  to  2on  in  diameter,  which  may  be  found  in  the  nerve-cells,  especially 
those  of  the  cotnu  ammonis  (Hippocampus  major). 

These  bodies  were  first  described  by  Negri  in  1903.  In  street  rabies  large  amoe- 
boid forms  from  18  to  23;*  may  be  found,  while  in  the  nerve  tissues  of  animals  with 
"fixed"  virus  only  minute  forms,  0.5/4  or  less,  may  be  detected.  The  fact  that  the 

362 


RABIES 


363 


virus  will  pass  through  a  Berkefeld  filter  is  no  argument  against  its  protozoal 
nature.  Calkins  considers  it  to  be  of  rhizopod  affinity.  The  term  Neuroryctes 
hydrophobias  has  been  given  it.  The  bodies  are  present  four  to  seven  days 
before  the  onset  of  symptoms.  They  may  be  demonstrated  by  staining  smears  of 
gray  brain  substance  by  some  Romanowsky  method,  especially  by  the  Giemsa  stain. 
The  smears  should  be  made  by  mashing  the  thin  slice  of  gray  matter  taken  from  i. 
Cornu  ammonis,  2.  Region  of  fissure  of  Rolando — in  dog  crucial  sulcus — or  3. 
Cerebellum,  with  a  cover-glass  against  the  slide.  Afterward  the  cover-glass  is  gently 
drawn  along  the  slide. 

The  smear  on  the  slide  is  then  fixed  in  methyl  alcohol  for  two  to  three  minutes, 
washed  with  water  and  covered  with  a  stain  made  by  adding  3  drops  of  Sat.  ale. 
sol.  of  basic  fuchsin  to  10  c.c.  of  distilled  water  and  then  adding  2  c.c.  of  Loffler's 
methylene  blue  solution.  The  stain  on  the  slide  is  then  steamed  gently  and  after- 
ward washed  with  water  and  dried. 


FIG.  105. — Two  nerve  cells  of  hippocampus  major  (smear  preparation)  showing 
Negri  bodies.  A,  Negri  bodies;  B,  inner  bodies  within  the  Negri  bodies.  (After 
Reichel,  American  Veterinary  Review.) 

As  their  relation  to  the  nerve-cell  is  more  or  less  disturbed  by  such  a  method 
it  is  preferable  to  fix  brain  tissue  from  the  region  of  the  cornu  ammonis  for  five  to 
seven  hours  in  Zenker's  fluid,  then  to  imbed  in  paraffin  and  make  sections.  These  are 
stained  with  Giemsa's  stain  and  the  Negri  bodies  are  brought  out  as  iliac-red  bodies 
in  the  blue  cytoplasm  of  the  nerve-cells.  It  is  necessary  to  differentiate  in  95% 
alcohol. 

In  the  Lentz  method  the  3;*  sections,  after  removal  of  the  paraffin,  are  flooded 
with  absolute  alcohol.  They  are  then  stained  with  a  1/2%  solution  of  eosin  in  60% 
alcohol  for  one  minute.  Wash  in  water  and  next  stain  for  one  minute  in  Loffler's 
methylene  blue.  Again  wash  in  water.  Apply  Lugol's  solution  to  the  section  for 
one  minute  and  then  differentiate  alternately  in  methyl  alcohol  and  water  until  the 
section  is  pink.  After  washing  in  water,  again  stain  with  Loffler's  blue  for  one-half 
a  minute,  then  wash  in  water  and  dry  carefully  with  filter-paper.  Now  differentiate 
in  alkaline  alcohol  (i  drop  of  a  5%  solution  NaOH  in  30  c.c.  absolute  alcohol)  until 


364  RABIES,  VACCINIA  AND   THE   FILTERABLE  VIRUSES 

the  section  is  pink,  then  quickly  differentiate  in  acid  alcohol  (i  drop  50%  acetic 
acid  in  30  c.c.  absolute  alcohol)  until  a  slight  blue  outline  to  the  ganglion  cells  is 
obtained.  Treat  rapidly  with  absolute  alcohol  and  xylol  and  mount  in  balsam. 
The  Negri  bodies  show  as  light  carmine  pink  bodies  on  the  light  blue  ground  of  the 
gangh'on  cells.  In  the  interior  of  the  pink  bodies  dark  blue  dots  or  rings  may  be 
observed. 

This  method  can  also  be  used  for  brain  smears. 

In  addition  to  examining  for  the  Negri  bodies,  a  rabbit  may  be  inoculated 
subdurally  with  a  sterile  salt-solution  emulsion  of  the  medulla  of  the  dead  dog. 

If  the  brain  and  medulla  of  the  dog  are  to  be  sent  to  a  laboratory 
for  examination  they  should  be  packed  in  ice  or  placed  in  glycerine. 
Take  of  glycerine  one  part  and  one  part  water.  Sterilize  the  diluted 
glycerine  by  boiling,  allow  to  cool,  and  drop  the  pieces  of  brain  tissue 
into  this.  This  does  not  kill  the  virus. 

When  from  advanced  putrefaction,  or  otherwise,  the  Negri  bodies  cannot  be 
found  the  changes  in  the  Gasserian  ganglia  may  give  a  diagnosis.  In  typical  lesions 
the  ganglion  cells  are  more  or  less  completely  destroyed  and  replaced  by  cells  of 
other  types. 

When  a  person  is  bitten  by  a  dog  suspected  of  being  rabid  the  following 
simple  measures  should  be  instituted.  The  dog  should  be  kept  under  observation 
in  a  safe  quiet  place  and  will  show  clinical  evidence  of  rabies  within  five  days  and 
will  die  shortly  afterward  in  case  rabies  exists.  When  the  animal  dies  the  head 
and  several  inches  of  the  neck  should  be  removed  and  packed  in  ice  and  sent  to  the 
nearest  laboratory. 

Antirabic  serum  has  been  prepared  by  injecting  sheep  with  emulsions  of  rabbits' 
cord  and  brain — at  first  intravenously,  then  subcutaneously. 

The  thorough  cauterization  of  the  dog-bite  wound  with  pure  nitric 
acid,  as  soon  as  possible  after  the  bite,  is  imperative  even  when  the  Pas- 
teur treatment  can  be  given  later. 

VACCINIA  is  a  disease  produced  artifically  by  the  injection  of  vaccine  virus  ob- 
tained from  the  calf.  The  material  for  vaccine  is  taken  from  vesicles  about 
one  week  after  the  inoculation.  The  most  potent  material  is  in  the  pulp  at  the  base 
of  the  vesicle  and  not  in  the  lymph  which  exudes  from  the  vesicle.  The  pulp  is 
ground  up  and  mixed  with  an  equal  amount  of  glycerine,  which  acts  not  only  as  a 
preservative  but  as  a  mild  antiseptic  for  nonsporing  bacteria.  The  calves  are  autop- 
sied  after  the  pulp  has  been  curetted  from  the  inoculated  skin  of  the  abdomen  to  be 
sure  that  no  disease  exists  in  the  calves.  The  virus  is  afterward  tested  for  pus  organ- 
'  isms,  tetanus,  and  foot  and  mouth  disease. 

Guarnieri  in  1892  first  observed  small  bodies  near  the  nucleus  of  infected  epi- 
thelial cells,  He  called  them  Cytoryctes  vacciniae.  Calkins  regards  these  bodies 
as  well  as  the  Negri  bodies  as  being  rhizopods  and  the  distributed  chromatin  as 
idiochromidia  (granules  of  nuclear  chromatin  within  the  cytoplasm). 


RABIES  365 

THE  FILTERABLE  VIRUSES. 

The  first  disease  of  which  the  virus  was  found  to  be  capable  of  pass- 
ing through  the  finest  porcelain  filter  was  that  of  foot  and  mouth  disease 
(LofBer  and  Frosch,  1898). 

The  filter  which  is  ordinarily  used  for  testing  for  the  passage  of  such 
disease  agents  is  the  Berkefeld  filter,  one  made  of  diatomaceous  earth. 
Of  the  infections  belonging  to  man  in  which  such  a  passage  of  blood  or 
serum  through  the  pores  of  a  porcelain  filter,  capable  of  keeping  back 
even  such  a  small  bacterial  organism  as  that  of  Malta  fever,  but  which 
does  not  hold  back  their  virus,  we  have  the  following:  foot  and  mouth 
disease,  trachoma,  molluscum  contagiosum,  vaccinia,  variola,  rabies, 
typhus  fever,  measles,  scarlet  fever,  yellow  fever,  dengue,  Papataci 
fever  and  poliomyelitis. 

There  are  many  diseases  of  this  nature  which  are  important  among  the  domes- 
ticated animals,  such  as  pleuropneumonia  of  cattle,  African  horse  sickness  and  hog 
cholera.  The  viruses  of  pleuropneumonia  of  cattle  and  poliomyelitis  have  been 
obtained  in  artificial  cultures.  Some  of  these  viruses  seem  related  to  bacterial 
infections  and  others  to  protozoal  ones.  These  viruses  differ  as  to  method  of  trans- 
mission, pleuropneumonia  of  cattle  being  transmitted  by  inhalation,  rabies  and 
vaccinia  by  the  cutaneous  atrium,  hog  cholera  by  ingestion  and  many  of  those  sup- 
posed to  have  protozoal  affinities,  as  yellow  fever,  Papataci  fever  and  horse  sickness 
by  mosquitoes. 

As  a  rule  these  viruses  are  destroyed  by  a  temperature  of  55°  C.  in  a  few  minutes. 


MISCELLANEOUS  NOTES. 


MISCELLANEOUS  NOTES. 


MISCELLANEOUS  NOTES. 


MISCELLANEOUS  NOTES. 


MISCELLANEOUS  NOTES. 


APPENDIX. 


A— PREPARATION  OF  TISSUES  FOR  EXAMINATION  IN 
MICROSCOPIC  SECTIONS. 

i.  Fixation: 

a.  It  is  most  important  that  the  tissues  to  be  examined  be  placed  in  the  fixing 
fluid  as  soon  after  death  or  operation  as  possible.     Degenerative  changes  are  in  this 
way  avoided. 

b.  The  piece  of  tissue  to  be  fixed  must  not  be  too  large.     Using  a  sharp  scalpel, 
or  preferably  a  razor,  a  slab  of  tissue  about  one-half  an  inch  square  and  not  more 
than  one-fifth  of  an  inch  thick  should  be  dropped  into  the  bottle  containing  the  fixa- 
tive.    The  bottom  of  this  bottle  should  have  a  thin  layer  of  cotton  with  a  piece  of 
filter-paper  covering  it.     There  should  be  at  least  twenty  times  as  great  a  volume 
of  fixing  fluid  as  of  tissue  to  be  fixed.     Delicate  tissues,  as  pieces  of  gut,  should  be 
attached  to  pieces  of  glass,  wood,  cardboard,  or  blotting  paper  before  being  placed  in 
the  fixative. 

c.  The  most  convenient  fixative  for  the  average  medical  man  is  a  10%  solution 
of  ordinary  commercial  formalin  (4%  of  formic  aldehyde  gas),  either  in  water  or, 
preferably,  in  normal  salt  solution.     Fixation  is  complete  in  from  twelve  to  twenty- 
four  hours.     By  placing  in  the  incubator,  at  37°  C.,  two  to  twelve  hours  in  the  forma- 
lin solution  suffices.     If  fixed  in  the  paraffin  oven  (56°  C.),  fixation  is  accomplished 
in  about  one-half  hour. 

Formalin  once  used  for  fixation  must  be  thrown  away. 

The  fixative  which  probably  gives  the  best  histological  pictures  and  with  which 
we  obtain  the  most  satisfactory  haematoxylin  staining  is  Zenker's  fluid.  This  is 
Miiller's  fluid  containing  5%  of  corrosive  sublimate.  It  also  contains  5%  of  glacial 
acetic  acid,  which  latter  is  only  added  just  before  we  are  ready  to  fix  the  piece  of 
tissue.  Miiller's  fluid  is: 

Pot.  bichromate,  2 .  5  grams. 

Sod.  sulphate,  i .  o  grams. 

Water,  100.0  c.c. 

Zenker's  fluid  fixes  in  about  twenty-four  hours.  After  all  corrosive  sublimate 
fixatives  we  should  wash  the  tissues  in  running  water  for  twelve  to  twenty-four 
hours.  The  precipitate  of  mercury  in  the  tissues  is  best  gotten  rid  of  by  treating  the 
section  on  the  slide  with  Lugol's  solution,  rather  than  the  tissue  in  bulk  with  iodine 
alcohol. 

In  Orth's  fluid  we  add  10%  of  formalin  to  Miiller's  fluid  (recommended  for  nerve 
tissue). 

371 


372  APPENDIX 

A  saturated  corrosive  sublimate  solution  in  salt  solution  with  the  addition  of  5% 
of  glacial  acetic  acid  may  be  used  as  a  substitute  for  Zenker's  fluid. 

2.  Dehydration. — After  washing  for  twelve  to  twenty-four  hours  in  running 
water,  following  corrosive  sublimate  fixation,  or  simply  washing  for  a  few  minutes 
after  formalin,  the  tissues  should  be  placed  in  70%  alcohol.     They  may  be  kept  in 
this  indefinitely.     If  they  are  to  be  sent  to  a  laboratory  for  sectioning,  it  is  advisable 
to  moisten  a  pledget  of  cotton  in  70%  alcohol  and  fill  in  the  bottom  of  the  bottle  with 
it.     Then  drop  in  the  tissues  and  pack  in  gently  over  them  sufficient  70%  alcohol- 
saturated  cotton  to  fill  up  the  bottle.     All  the  alcohol  should  be  absorbed  by  the 
cotton  so  that  if  the  bottle  should  break  in  transit  there  would  be  no  damage  from  the 
alcohol.     The  stopper  of  the  bottle  should  be  paraffined  or  sealed  with  wax. 

Tissues  may  be  left  in  the  70%  alcohol  twelve  to  twenty-four  hours  and  should 
then  be  transferred  to  95%  alcohol  for  an  equal  time.  They  are  then  transferred  to 
absolute  alcohol,  where  they  remain  from  two  to  twelve  hours  and  are  then  placed  in 
xylol.  The  time  in  xylol  should  be  as  short  as  possible.  So  soon  as  the  tissue  looks 
clear  it  should  be  removed — thirty  minutes  to  two  hours. 

3.  Imbedding. — The  tissue  is  now  transferred  to  melted  paraffin.     Paraffin  melt- 
ing at  48°  C.  for  winter  work,  and  that  melting  at  54°  C.  for  summer  is  to  be  recom- 
mended.    The  time  in   the  paraffin  should  not  be  prolonged.     Two  hours  will 
ordinarily  suffice.     Some  leave  in  the  paraffin  for  twelve  to  twenty-four  hours. 

Next  take  a  paper  box  (made  of  stiff  writing-paper  folded  over  a  square  of  wood) 
and  fill  with  the  melted  paraffin.  As  quickly  as  possible  drop  in  the  piece  of  tissue 
taken  out  of  the  paraffin  bath  with  heated  forceps  and,  so  soon  as  the  paraffin  begins 
to  solidify  on  the  surface,  place  the  paper  box  in  ice  water.  When  paraffin  is  rapidly 
cooled,  crystallization  is  less. 

The  Acetone  Method. — Take  the  tissues  out  of  the  70%  alcohol  and  place  in  ace- 
tone. After  remaining  in  acetone  for  one  to  two  hours,  the  tissues  should  be  trans- 
ferred to  fresh  acetone  for  an  equal  length  of  time.  Dry  calcium  chloride  in  the  bot- 
tom of  the  acetone  bottles  keeps  it  dehydrated.  They  should  then  be  placed  in 
xylol  for  about  one-half  hour  and  then  embedded  in  paraffin  as  directed  above. 

The  Chloroform  Method. — The  procedure  may  be  the  same  as  in  the  method  of 
passing  through  alcohols  to  xylol,  substituting  chloroform  for  xylol  and  then  trans- 
ferring to  paraffin. 

Where  absolute  alcohol  is  not  obtainable,  very  satisfactory  results  may  be 
obtained  by  transferring  to  a  mixture  of  95%  alcohol  and  chloroform  after  immer- 
sion in  95%  alcohol.  Then  going  from  the  alcohol-chloroform  mixture  to  pure 
chloroform,  thence  to  paraffin. 

Rapid  paraffin  imbedding  methods. 

When  a  piece  of  tissue  is  not  more  than  one-fourth  inch  square  and  one-eighth 
inch  thick,  it  is  very  easy  to  run  it  through  in  three  to  six  hours.  Thus: 

10%  Formalin  (in  37°  C.  incubator),  i  hour. 

70%  Alcohol  (in  37°  C.  incubator),  i  hour. 

95%  Alcohol  (in  37°  C.  incubator),  i  hour. 

Absolute  Alcohol  (in  37°  C.  incubator),  1/2  hour. 

Xylol  (in  37°  C.  incubator),  1/2  hour. 

Paraffin  (in  55°  C.  incubator),  1/2  to  2  hours. 


APPENDIX  373 

Method  of  Lubarsch. — In  this  excellent  method  small  pieces  of  tissue  not  more 
than  1/5  inch  thick  are  placed  in  a  wide  test-tube  containing  10%  formalin  for  10  to 
15  minutes,  changing  the  fluid  twice.  Transfer  to  95%  alcohol  10  minutes  changing 
alcohol  once.  Absolute  alcohol,  for  10  minutes  changing  twice.  Pure  aniline  oil 
until  tissues  are  transparent,  15  to  30  minutes.  Xylol,  changing  two  to  three  times 
or  until  the  xylol  is  no  longer  yellow,  10  to  20  minutes.  Imbed  in  paraffin  for  20 
minutes  to  i  hour.  During  the  entire  process  keep  the  test-tube  in  a  water  bath 
or  incubator  at  50°  C.  It  is  preferable  to  have  a  good  microtome.  The  best  is  that 
of  Minot.  Very  satisfactory  sections  can  be  cut  with  the  various  types  of  student 
microtomes,  costing  from  $12  to  $20. 

(In  using  a  hand  microtome,  a  razor  with  a  flat  edge  is  necessary.  After  expe- 
rience, sections  thin  enough  for  histological  but  not  for  bacteriological  examination 
can  be  made.) 

If  the  piece  of  tissue  is  properly  dehydrated  and  imbedded,  thin  sections  (3  to 
io/0  should  be  easily  obtained,  provided  the  knife  be  sharp.  One  advantage  about 
the  paraffin  method  is  that  it  is  only  necessary  to  have  a  small  part  of  the  blade  in 
proper  condition.  With  celloidin  the  entire  cutting  edge  must  be  perfect.  Having 
cut  the  sections,  they  should  be  dropped  on  the  surface  of  a  bowl  of  warm  water 
(45°  C.).  This  causes  the  section  to  flatten  out  evenly. 

Decalcification. — This  is  best  accomplished  by  fixing  in  10%  formalin  for  twenty- 
four  hours,  then  placing  a  small  piece  of  the  bone  (not  exceeding  one-half  inch  square 
and  one-fifth  of  an  inch  thick)  in  concentrated  sulphurous  acid. 

This  decalcifies  in  about  2  to  7  days.  Wash  thoroughly  in  alkaline  water 
and  then  in  tap  water.  Pass  through  alcohols  and  xylol  and  imbed  and  section  as 
before  described. 

To  Stain  Sections. — It  is  first  necessary  to  affix  the  section  to  a  slide  or  cover-glass. 
To  attach  the  section  firmly  to  the  slide,  so  that  it  will  not  become  detached  in 
subsequent  treatment,  pick  up  a  section  on  a  strip  of  cigarette  paper. 

A  sheet  of  cigarette  paper  is  cut  into  about  five  pieces  (1/2X1  1/2  inches). 
Inserting  the  strip  of  cigarette  paper  under  the  section,  it  is  easily  lifted  up  out  of 
the  water.  Then  apply  the  slip  of  cigarette  paper,  section  downward,  to  a  perfectly 
clean  slide.  Blot  with  a  piece  of  filter-paper,  then  strip  off  the  piece  of  filter-paper 
leaving  the  section  smoothly  applied  to  the  slide.  Next  place  in  the  37°  C.  incubator 
for  twelve  to  twenty-four  hours  and  the  section  will  be  found  to  be  so  firmly  attached 
that  it  will  not  be  dislodged  by  subsequent  treatment. 

For  Immediate  Diagnosis. — Take  a  loopful  of  albumin  fixative  (white  of  fresh 
egg,  50  c.c.;  glycerin,  50  c.c.;  sodium  salicylate,  i  gram)  and  deposit  it  on  a  cover- 
glass.  Now  take  up  a  loopful  of  30%  alcohol  (i  drop  of  95%  alcohol  and  two  drops 
of  water)  and  applying  it  over  the  albumin  fixative,  smear  out  the  mixture  uniformly 
over  the  cover-glass. 

2.  Pick  up  a  section  on  a  strip  of  cigarette  paper  and  apply  it  to  the  prepared  sur- 
face on  the  cover-glass.     Blot  with  gentle  pressure  with  a  piece  of  filter-paper  over 
the  strip  of  cigarette  paper,  and  strip  off  this  latter,  leaving  the  section  attached  to 
the  cover-glass. 

3.  Now,  turning  the  flame  of  the  Bunsen  burner  down  very  low  or  with  a  small 
alcohol  flame,  we  hold  the  cover-glass  in  a  Stewart's  forceps,  section  side  up,  over 
the  flame  and  slowly  lower  it  until  the  paraffin  is  observed  to  melt.     This  shows  a 


374  APPENDIX 

temperature  of  about  50°  C.  The  section  is  fixed  by  the  coagulation  of  the  albumin 
at  about  70°  C.  To  obtain  this  temperature  lower  the  cover-glass  still  more,  and  the 
moment  vapor  is  seen  to  rise  from  the  section  it  indicates  the  attachment  of  the  sec- 
tion to  the  cover-glass. 

4.  Flood  section  on  cover-glass  or  slide  with  xylol;  this  dissolves  out  the  paraffin. 
It  is  better  to  pour  off  the  first  xylol  and  drop  on  fresh  xylol  (one  minute). 

5.  Remove  xylol  with  two  applications  of  absolute  alcohol  (one  minute). 

6.  Treat  specimen  with  two  or  three  applications  of  95%  alcohol  (one  to  two 
minutes). 

7.  Next  wash  in  water  (one  to  two  minutes). 

8.  Flood  specimen  with  hasmalum  or  Delafield's  haematoxylin  (three  to  seven 
miautes). 

9.  Wash  in  tap  water  for  about  two  to  five  minutes  until  a  purplish  tinge  is  devel- 
oped in  the  section.     The  alkali  in  ordinary  tap  water  develops  this  color. 

10.  Apply  i  to  1000  eosin  for  thirty  seconds  to  one  minute. 

11.  Wash  in  water;  then  in  95%  alcohol;  then  in  absolute  alcohol. 

12.  Apply  a  few  drops  of  xylol  and  as  soon  as  the  section  is  perfectly  transparent 
mount  in  balsam. 

The  staining  by  haematoxylin  and  eosin  is  the  best  for  the  study  of  the  histology 
of  a  section.  It  only  requires  about  ten  minutes  to  run  a  preparation  through  for 
diagnosis  by  this  method. 

The  reagents  are  best  kept  in  dropping-bottles. 

The  staining  of  sections  on  slides  is  exactly  as  for  those  on  cover-glasses.  Cop- 
lin's  staining  jars  are  very  convenient  for  use  in  staining  slides. 

Where  the  cover-glass  method  is  used,  staining  by  Gram's  method,  acid-fast  stain- 
ing, capsule  staining,  etc.,  may  he  carried  out  as  for  bacterial  preparations. 

For  staining  Gram  positive  bacteria  in  sections,  the  Gram  method  as  for  bacterial 
preparations,  using  dilute  carbol  fuchsin  as  a  counterstain,  gives  good  results. 

For  Gram  negative  bacteria  stain  with  thionin  as  for  blood  preparations  (ten  to 
twenty  minutes).  Then  differentiate  in  i  to  500  acetic  acid  solution  for  ten  to  twenty 
seconds,  wash  with  water,  then  with  95%  alcohol,  and  quickly  through  absolute 
alcohol  and  xylol. 

Nicolle's  Method. — i.  Stain  with  Lo  frier's  methylene  blue  ten  to  fifteen  minutes. 

2.  Differentiate  in  i  to  500  acetic  acid  ten  to  twenty  seconds. 

3.  Place  in  i%  solution  of  tannin  for  a  few  seconds  (fixes  color). 

4.  Wash  in  water,  then  into  95%  alcohol,  absolute  alcohol,  xylol,  and  balsam. 

WEIGERT'S  IRON  H^MATOXYLIN. 

Solution  I. 

Haematoxylin,  i  gram. 

Alcohol  (95%),  100  c.c. 

This  must  be  allowed  to  ripen  for  some  days  and  does  not  keep  over  six  months. 

Solution  II. 

Liq.  Ferri  sesquichlor.  sp.  gr.  1.124  (about  10%)  4  c.c. 

HC1,  i  c.c. 

Water,  100  c.c. 


APPENDIX  375 

Mix  equal  parts  of  number  one  and  number  two.  The  mixture  only  keeps  about 
three  days.  The  HC1  prevents  overstaining. 

This  stain  followed  by  Van  Giesen's  stain  gives  more  perfect  results  than  any 
common  method  of  staining.  The  iron  haematoxylin  intensifies  the  sharpness  of 
the  Van  Geisen  differentiation. 

Van  Giesen's  Stain. — Take  of  i%  aqueous  solution  acid  fuchsin  from  5  to  15 
c.c.  Saturated  aqueous  solution  picric  acid  100  c.c.  The  method  of  using  is  to  first 
stain  with  haematoxylin  in  the  usual  way.  Then  pour  on  the  picric-acid  fuchsin 
solution  and  allow  to  stain  for  one  to  five  minutes.  Wash,  pass  through  alcohols 
and  xylol  and  mount  in  balsam. 

Connective-tissue  fibers,  axis  cylinders,  and  ganglion  cells  are  stained  a  bright 
garnet  red.  Myelin,  muscle  fibers,  and  cells  generally  are  stained  yellow.  Nuclear 
staining  is  that  of  hasmatoxylin.  The  stronger  stain  is  used  for  nerve  tissue;  the 
weaker,  for  demonstrating  connective  tissue  in  tumors. 

Levaditi's  Method. — Take  small  pieces  of  tissue,  about  2  mm.  in  thickness,  and 
harden  in  10%  formalin  for  twenty-four  hours  and  then  in  alcohol  for  the  same  period; 
then  wash  in  water  for  a  short  period.  They  are  stained  in  a  freshly  made  solution 
of  silver  nitrate  1.5%  for  three  successive  days,  changing  the  solution  each  day, 
maintaining  the  blood  temperature,  and  excluding  light.  The  tissue  is  then  placed 
in  a  2%  solution  of  pyrogallic  acid,  with  the  addition  of  5%  formalin.  After  remain- 
ing in  this  for  twenty-four  hours,  light  being  excluded,  they  are  passed  through 
85%,  95%,  and  absolute  alcohol,  respectively;  embedded  in  paraffin;  and  cut  in 
about  5  micron  sections.  Equally  good  results  may  be  obtained  by  allowing  the 
silver  nitrate  to  act  at  room  temperature  and  embedding  in  celloidin. 

Romanowsky. — Staining  sections  with  Romanowsky  stains  is  not  very  satis- 
factory. The  differential  staining  seems  to  fade  out  in  passing  through  the  alcohols. 
This  may  be  avoided  by  blotting  the  section  after  staining  and  differentiation  and 
then  applying  the  xylol  to  the  blotted  section.  After  staining  with  Giemsa's  stain 
for  ten  to  fifteen  minutes,  differentiate  with  i  to  500  acetic  acid.  When  the  section 
has  a  pinkish  tinge,  wash  in  water,  dry,  clear  in  xylol,  and  mount. 

Good  tissue  staining  may  be  gotten  with  Wright's  stain.  After  removing  the 
paraffin  with  xylol  and  the  rylol  with  absolute  alcohol,  pour  on  a  sufficient  number  of 
drops  of  stain  and  immediately  dilute  with  an  equal  number  of  drops  of  water. 
Allow  the  diluted  stain  to  remain  for  three  to  five  minutes.  Next  wash  in  water, 
differentiate,  until  the  tissue  has  a  pinkish  tinge,  in  i  to  500  acetic  acid.  This 
differentiation  is  best  done  in  a  tumbler  of  the  dilute  acetic  acid. 

After  washing  in  water,  quickly  pass  through  95%  and  absolute  alcohol,  clear  in 
xylol,  and  mount. 

Skin  Sectioning. — Of  all  tissues  that  of  skin  offers  the  greatest  difficulty  in  pre- 
paring sections.  The  best  results  can  probably  be  obtained  by  fixation  in  picro- 
sublimate  (saturated  aqueous  solution  picric  acid  i  part;  saturated  aqueous  solu- 
tion bichloride  of  mercury  one  part);  to  this  stock  mixture  add  5%  glacial  acetic 
acid  just  before  using.  Fix  small  pieces  of  skin  six  to  eighteen  hours.  Transfer 
direct  to  70%  alcohol  in  which  the  tissue  may  be  kept  indefinitely. 

For  sectioning  run  through  alcohols  to  absolute  and  then  to  a  mixture  of  absolute 
alcohol  and  carbon  bisulphide  (equal  parts).  Leave  until  tissue  sinks,  then  transfer 
to  pure  carbon  bisulphide  until  tissue  sinks.  Then  transfer  to  a  saturated 


376  APPENDIX 

solution  of  paraffin  in  carbon  bisulphide  and  thence  to  paraffin.  Bisulphide  of 
carbon  has  the  disadvantage  of  foul  odor  and  inflammability  but  does  not  seem  to 
render  tissues  brittle  and  difficult  to  section  as  does  xylol. 

NEUROLOGICAL  STAINING  METHODS. 

Neuropathology  practically  dates  from  the  introduction  of  Marchi's  method  of 
staining  in  1885. 

Ordinary  osmic  acid  stains  both  normal  and  pathological  fat.  With  Marchi's 
method  only  the  oleic  acid  of  fatty  degeneration  is  stained. 

The  method  is  not  useful  until  three  or  four  days  have  elapsed  from  the  onset 
of  the  condition  causing  the  degeneration  and  it  is  applicable  for  only  three  or  four 
months  because  by  that  time  phagocytes  have  taken  up  the  pathological  fat  which 
is  stained  in  the  Marchi  method.  The  Weigert  method  is  the  one  to  use  after  a 
period  of  three  or  four  months.  In  Weigert's  stain  only  the  normal  myelin  sheath 
is  stained  and  the  lack  of  staining  of  myelin  sheaths  in  degenerated  areas  is  the  basis 
of  the  stain.  For  demonstrating  axonal  reactions  or  other  degenerative  changes 
in  nerve  cells,  as  shown  by  bulging  of  the  concave  sides  of  the  cells,  eccentric  nucleus 
and  granular  appearance  of  the  tigroid  bodies,  Nissl's  method  is  the  best. 

For  neuroglia  fiber  staining  Mallory's  phosphotungstic  acid  haematoxylin  is  to 
be  recommended. 

I.  For  Marchi's  Method. — Small  pieces  of  nerve  tissue  are  hardened  in  Miiller's 
fluid  for  seven  to  ten  days  and  are  then  transferred  to  a  mixture  of  two  parts  Miiller's 
fluid  and  one  part  of  a  i%  osmic  acid  solution  and  should  remain  in  this  mixture 
for  about  seven  days.     The  tissue  thus  treated  is  run  through  alcohols  and  imbedded 
in  paraffin  in  the  usual  way. 

II.  For  Weigert-Pal  Method. — Thin  slices  of  tissue  are  fixed  in  10%  formalin 
in  about  four  days.     The  tissue  should  then  be  transferred  to  5%  potassium  bichro- 
mate for  about  twelve  days.     The  tissue  is  then  imbedded  and  sections  cut.     If 
only  recently  mordanted  these  sections  may  be  at  once  stained  with  Weigert's 
haematoxylin  for  twelve  to  twenty-four  hours  (10  c.c.  ripened  10%  solution  haema- 
toxylin in  absolute  alcohol  and  90  c.c.  water).     Wash  in  water  to  which  about  2% 
of  a  saturated  solution  of  lithium  carbonate  has  been  added.     Now  differentiate 
from  one-half  to  five  minutes  in  1/4%  solution  of  potassium  permanganate  until 
the  gray  matter  looks  a  brownish-yellow.     Next  treat  sections  with  oxalic  acid 
i  gram,  potassium  sulphate  i  gram  and  water  200  c.c.  until  the  gray  matter  is 
almost  colorless.     This  takes  only  a  few  seconds.     Wash  in  water,  pass  through 
alcohols  and  xylol  and  mount  in  balsam. 

III.  For  Nissl  staining  either  thionin  or  Giemsa  staining  is  satisfactory. 

IV.  For  neuroglia  fiber  staining  use  Mallory's  Phosphotungstic  acid  haematoxylin. 

Take  of  haematein  ammonium,  o.i  gram. 

Water,  100.0  c.c. 

Phosphotungstic  acid  crystals  (Merck)  2.0  grams. 

Dissolve  the  haematein  in  a  little  water  with  the  aid  of  heat,  and  add  it  after  it  is 
cool  to  the  rest  of  the  solution;  no  preservative  is  required.  If  the  solution  stains 
weakly  at  first,  it  may  be  ripened  by  the  addition  of  5  c.c.  of  a  1/4%  aqueous 


APPENDIX  377 

solution  of  potassium  permanganate,  or  it  may  be  allowed  to  stand  for  a  few  weeks 
until  it  ripens  spontaneously. 

MAKING  AND  STAINING  OF  FROZEN  SECTIONS. 

The  various  types  of  ether  freezing  microtomes  are  not  very  satisfactory  when 
only  used  occasionally.  With  the  general  introduction  of  cylinders  containing 
compressed  carbon  dioxide,  which  is  used  for  aerating  waters,  we  have  at  hand  a 
practical  and  convenient  method  of  making  frozen  sections. 

The  instrument  makers  furnish  a  freezing  microtome  of  the  Bardeen  type  which 
can  be  attached  directly  to  the  cylinder  by  a  revolving  clamp  nut. 

It  is  necessary  to  have  a  stand  to  support  the  iron  cylinder  in  a  horizontal  posi- 
tion The  tissue,  which  may  be  taken  at  operation  for  immediate  diagnosis,  or 
which  preferably  has  been  fixed  in  formalin  for  twelve  to  eighteen  hours  is  immedi- 
ately placed  in  water.  If  the  tissues  have  been  in  alcohol  it  will  require  hours  of 
washing  before  they  can  be  frozen.  The  piece  of  tissue  which  is  to  be  frozen  should 
not  be  more  than  one-fifth  of  an  inch  thick.  Having  placed  the  piece  of  tissue  on 
the  freezing  box  of  the  microtome  we  turn  the  valve  of  the  cylinder  to  allow  the 
gradual  escape  of  gas.  When  frozen  solid,  we  elevate  the  freezing  box  holding  the 
frozen  tissue,  by  revolving  a  graduated  disc  with  the  left  hand.  In  the  right  hand 
we  firmly  grasp  a  well-sharpened  blade  of  a  carpenter's  plane  mounted  in  a  wooden 
handle.  This  is  held  at  an  angle  of  45  degrees  to  the  polished  ways  of  the  micro- 
tome. By  alternate  shoving  and  withdrawing  of  the  blade,  held  rigidly,  we  accumu- 
late on  the  blade  a  number  of  sections.  Then  dip  the  blade  in  a  vessel  of  water  to 
detach  the  sections  which  float  in  the  water.  Keep  repeating  the  process  until 
numerous  satisfactory  sections  are  obtained.  Handles  for  holding  the  Gillette 
razor  blades  are  good  substitutes  for  the  carpenter's  plane. 

These  sections  may  be  picked  up  with  a  strip  of  cigarette  paper  and  applied  to  a 
clean  slide  upon  which  a  very  small  loopful  of  albumin  fixative  has  been  smeared 
out  with  30%  alcohol.  The  piece  of  cigarette  paper  with  the  section  underneath 
is  then  firmly  smoothed  out  upon  the  slide  with  filter-paper.  The  piece  of  cigarette 
paper  is  then  carefully  stripped  off  and  the  section  remains  attached  to  the  slide. 
By  careful  heating  over  a  very  small  flame,  until  the  vapor  just  arises,  the  section 
is  fixed  to  the  slide  and  we  can  then  stain  the  section  in  any  way  that  may  be  desired. 

NOTE. — The  procedures  for  carrying  the  tissues  through  celloidin  are  not  given 
as  it  requires  perfect  condition  of  the  entire  cutting  surface  of  the  microtome  knife 
and  a  considerable  time  for  the  passage  through  regents  and  celloidin.  It  is  more 
suitable  as  a  method  when  sections  for  class  work  are  to  be  prepared. 

B— MOUNTING  AND  PRESERVATION  OF  PATHOLOGICAL  SPECIMENS 
AND  ANIMAL  PARASITES. 

To  Mount  Small  Round  Worms. — Wash  the  hook,  whip,  or  filarial  worm  in  salt 
solution,  then  drop  in  70%  alcohol  containing  5%  of  glycerine;  the  glycerine-alcohol 
mixture  being  at  a  temperature  of  60°  C.  When  cool,  pour  into  Petri  dishes  and 
allow  the  alcohol  to  evaporate  in  the  37°  C.  incubator. 

Mount  in  glycerine  jelly,  preferably  in  a  concave  slide,  and  ring  the  preparation 


378  APPENDIX 

with  gold  size.  The  following  is  the  formula  for  Kaiser's  glycerine  jelly:  Soak  one 
part  of  gelatin  in  six  parts  of  distilled  water  for  two  hours.  Then  add  seven  parts 
of  glycerine.  To  the  mixture  add  i  %  of  carbolic  acid,  warm  for  fifteen  minutes, 
with  constant  stirring,  and  then  filter  through  cotton. 

To  Prepare  Tape -worms. — Wash  in  salt  solution.  Wrap  around  a  piece  of  glass 
as  a  glass  slide  and  fix  in  salt  solution  containing  2  to  5%  of  formalin.  Then  keep 
the  preparation  permanently  in  70%  alcohol.  If  preferred,  the  specimen  may  be 
run  through  alcohols  and  xylol  and  mounted  in  balsam. 

Larvae. — Mosquito  larvae  may  either  be  prepared  as  for  small  round  worms  or 
they  may  be  dropped  into  70%  alcohol  at  60°  C.  and  then  passed  through  alcohols 
and  cleared  in  xylol  and  mounted  in  balsam.  Flukes  and  insects  may  require  treat- 
ment with  hot  (60°  to  70°  C.)  solution  of  10  to  20%  sodium  hydrate  solution.  Then 
wash  thoroughly  in  water  and  subsequently  pass  through  alcohols  to  xylol  and  mount 
in  balsam.  Clove  oil  or  cedar  oil  clears  more  slowly,  but  makes  specimens  less 
brittle  than  does  xylol.  Another  satisfactory  method  is  to  drop  insects  or  larvae 
into  acetone  at  60°  C.  and  after  being  in  this  from  one  to  twelve  hours  to  clear  in 
xylol  or  clove  oil  and  mount  in  balsam. 

^  Nematodes. — Looss  has  a  method  of  first  washing  a  small  nematode  or  delicate 
fluke  in  salt  solution.  Then  pouring  this  first  salt  solution  out  of  the  test-tube  in  which 
the  washing  was  carried  out,  to  add  fresh  salt  solution,  and  then  an  equal  amount 
of  saturated  aqueous  solution  of  bichloride  of  mercury.  The  shaking  is  easily 
carried  on  in  the  test-tube.  After  washing  in  water  the  worm  is  passed  through 
alcohols,  one  strength  of  which  should  contain  iodine.  Clear  in  xylol  and  mount  in 
balsam. 

An  excellent  method  is  that  of  Langeron. 

After  washing  in  salt  solution  fix  for  a  few  hours  in  5%  formalin.  Then  transfer 
to  lactophenol  which  has  been  diluted  with  an  equal  amount  of  water.  Allow  to 
remain  in  this  solution  for  several  hours  and  then  transfer  to  pure  lactophenol  in 
which  fluid  the  specimens  are  to  be  mounted.  Ring  with  paraffin  or  with  gold  size. 
(To  make  lactophenol  take  two  parts  of  glycerine  and  one  part  each  of  distilled 
water,  crystallized  carbolic  acid  and  lactic  acid.) 

A  quick  method  of  preparing  small  nematodes  for  examination  is  to  fix  them  for 
from  two  to  twelve  hours  in  5  to  10%  formalin,  this  being  heated  to  60°  C.  at  the 
time  the  worms  are  dropped  into  it.  Then  transfer  to  the  following  solution: 

Glucose  syrup  (glucose,  48;  water,  52),  100  c.c. 

Methyl  alcohol,  20  c.c. 

Glycerine,  10  c.c. 
Camphor,  q.s.  (a  small  lump  for  preservation). 

They  may  be  mounted  directly  in  this  and  the  cover-slip  ringed  with  about  60°  C. 
paraffin,  followed  with  gold  size. 

Preparations  so  cleared  and  mounted  in  glycerine  jelly  should  also  be  ringed  with 
paraffin  or  °ome  cement. 

Flukes,  cestodes,  and  nematodes  are  best  stained  with  carmine.  The  following 
is  a  good  formula. 

Dissolve,  by  boiling,  4  grm.  carmine  in  thirty  drops  HC1  and  15  c.c.  water.     Then 


APPENDIX  379 

add  95  c.c.  of  85%  alcohol  and  filter  while  hot.  Neutralize  with  ammonia  until 
precipitate  begins  to  form.  Then  filter  cold. 

i.  Stain  parasites  taken  from  70%  alcohol  for  five  to  twenty  minutes.  2.  Dif- 
ferentiate in  3%  hydrochloric  acid.  3.  Pass  through  alcohols  to  xylol  and  mount  in 
balsam. 

Mites,  Fleas  and  Various  Small  Insects. — By  simply  taking  one  or  two  drops 
of  liquid  petrolatum  and  mounting  the  specimen  in  it  then  covering  with  a  cover- 
glass  one  is  able  to  study  the  details  of  these  objects  almost  as  well  as  if  they  were 
passed  through  acetone  and  xylol  into  balsam.  Liquid  petrolatum  is  also  most 
excellent  for  mounting  the  aerial  hyphas  of  fungi  with  their  sporangia  as  well  as  for 
Romanowsky  stained  blood  smears. 

Pathological  tissues  which  are  to  be  sent  to  a  laboratory  for  sectioning  or  to  be 
kept  for  future  study  should  be  fixed  by  one  of  the  methods  given  in  Section  A  of 
the  appendix. 

Formalin  fixation  is  the  more  convenient — that  with  Zenker's  fluid  the  more 
perfect.  After  fixation  with  Zenker's  fluid  the  pieces  of  tissue  must  be  washed  in 
running  water  over  night. 

After  fixation  the  pieces  of  tissue  are  transferred  to  70%  alcohol  in  which  they  may 
be  kept  indefinitely. 

For  preservation  of  gross  specimens  the  method  KAISERLING  is  generally  used. 

Fix  for  from  one  to  five  days  in  Solution  I. 

Solution  I. 

Formaldehyde,  200  c.c. 

Water,  1000  c.c. 

Nitrate  of  potassium,  15  grams. 

Acetate  of  potassium,  30  grams. 

The  position  of  the  specimen  should  be  changed  from  day  to  day.  There  must 
be  at  least  five  times  as  much  fluid  as  specimen.  Drain  and  transfer  to  80%  alcohol 
for  a  few  hours,  then  into  95%  alcohol  until  the  natural  color  is  just  restored. 

Finally  preserve  in 

Acetate  of  potassium,  200  grams. 

Glycerine,  400  c.c. 

Water,  2000  c.c. 

It  is  advisable  to  keep  these  specimens  in  the  dark  as  light  destroys  the  natural 
color. 

To  Prepare  Flies  or  Mosquitoes  for  Transmission  Through  the  Mails. — Wrap 
the  insect  carefully  in  a  piece  of  tissue  paper  (toilet-paper  answers).  Impregnate 
sawdust  with  5%  carbolic  acid  solution  and  fill  around  the  tissue  paper  in  the 
box  containing  them.  (Barely  moisten.) 

It  is  very  satisfactory  to  take  a  tube  form  vial  with  a  cork  from  the  inner  sur- 
face of  which  two  small  shallow  holes  have  been  bored,  one  containing  parafor- 
maldehyd,  the  other  camphor.  The  insect  is  mounted  upon  a  pin  stuck  in  the 
cork,  which  latter  is  inserted  and  parafined  externally. 


380  APPENDIX 

C— PREPARATION  OF  NORMAL  SOLUTIONS. 

A  normal  solution  is  one  which  contains  the  hydrogen  equivalent  of  an  element, 
expressed  in  grams,  dissolved  in  sufficient  distilled  water  to  make  1000  c.c.  The 
hydrogen  equivalent  is  the  atomic  weight  of  any  element  divided  by  its  valence.  In 
a  base,  salt  or  acid,  we  use  the  molecular  weight  in  grams  divided  by  valence. 

What  may  be  considered  as  the  valence  of  a  base  is  shown  by  the  number  of 
hydroxyls  combined  with  it;  that  of  an  acid  by  the  number  of  replaceable  hydrogen 
atoms  which  it  contains. 

To  make  a  normal  solution,  dissolve  in  distilled  water  a  weight  in  grams  equal 
to  the  sum  of  the  atomic  weights  of  the  substance  divided  by  its  valence,  and  make 
up  the  volume  to  exactly  1000  c.c. 

NaOH  is  univalent.  Na  =  23.  O  =  i6.  H  =  i.  Dissolve  40  grams  NaOH 
in  water  and  make  up  to  exactly  1000  c.c. 

Oxalic  acid  is  COOH  — COOH+2H2O  which  gives  it  a  molecular  weight  of  126. 
As  it  contains  two  carboxyl  groups  it  is  dibasic,  and  it  is  necessary  to  divide  the 
molecular  weight  by  2,  so  that  for  a  normal  solution  of  oxalic  acid  we  dissolve  63 
grams  in  a  volume  of  distilled  water  made  up  to  1000  c.c. 

If  a  chemical  laboratory  is  not  accessible  one  may  prepare  normal  solutions  with 
an  error  so  slight  as  to  be  unimportant  in  clinical  work  in  the  following  way: 

Sodium  hydrate  being  very  hygroscopic,  it  is  impossible  to  accurately  prepare  a 
normal  solution  by  directly  weighing  out  the  substance.  Instead,  select  perfect 
crystals  of  oxalic  acid,  such  as  can  be  obtained  in  a  drug  store,  and  weigh  out  on  the 
most  accurate  apothecary  scales  obtainable  exactly  6.3  grams  of  the  most  perfect 
crystals  in  the  bottle.  Put  these  preferably  in  a  volumetric  flask  and  make  up  with 
distilled  water  to  1000  c.c.  Less  accurate  is  the  use  of  a  measuring  cylinder.  If 
care  is  used  this  should  give  N/io  solution  of  oxalic  acid  in  which  the  error  is  less 
than  i%. 

Having  N/io  acid  at  hand,  we  may  prepare  N/io  NaOH  in  the  following  way: 
Weigh  out  an  excess  of  sodium  hydrate  (5  grams  of  stick  caustic  soda)  and  dissolve 
in  1 100  c.c.  of  distilled  water.  Take  up  10  c.c.  of  this  solution  with  a  pipette  and  let 
it  run  into  a  beaker.  Add  six  drops  of  phenolphthalein  solution.  This  gives  a  violet- 
pink  color.  Fill  the  burette  with  the  N/io  oxalic  acid  solution  and  let  it  run  into 
the  sodium  hydrate  solution  in  the  beaker  until  the  pink  is  just  discharged.  Reading 
off  the  number  of  c.c.  of  the  N/io  acid  used,  we  know  the  strength  of  the  sodium 
hydrate  solution.  It  is  well  to  repeat  the  titration  and  take  an  average. 

If  10.5  c.c.  of  the  oxalic  acid  solution  were  required  it  would  show  that  the  sodium 
hydrate  solution  was  stronger  than  N/io,  as  only  10  c.c.  would  have  been  necessary 
if  the  NaOH  solution  had  been  N/io.  It  is  therefore  necessary  to  dilute  the  sodium- 
hydrate  solution  in  the  proportion  of  10  to  10.5.  Measure  exactly  1000  c.c.  of  the 
too  concentrated  sodium-hydrate  solution  and  add  to  it  50  c.c.  of  distilled  water,  mix 
thoroughly,  and  we  have  1050  c.c.  of  N/io  solution  of  NaOH.  ioooX  10.5  =  10,500. 
10,500-7-10=  1050. 

.  As  Acidum  hydrochloricum  U.  S.  P.  is  about  two-thirds  water  (68.1%)  to  make 
N/io  HC1,  which  would  require  3.65  in  1000  c.c.,  it  would  be  necessary  to  take  about 
three  times  this  amount  of  U.  S.  P.  acid.  Take  12  c.c.  of  the  acid  and  add  distilled 
water  to  make  uoo  c.c.  Put  10  c.c.  of  this  dilute  solution  in  a  beaker.  Add  phenol- 


APPENDIX  381 

phthalein  solution  and  titrate.  If  n  c.c.  of  N/io  NaOH  were  required  it  would  be 
necessary  to  add  100  c.c.  of  water  to  a  volume  of  1000  c.c.  of  the  diluted  hydrochloric 
acid.  1000X11  =  nooo-f-io=  noo. 

Other  acid  and  alkali  solutions  can  be  made  as  for  N/io  HC1  and  N/io  NaOH. 


D— DISEASES  OF  UNKNOWN  OR  NOT  DEFINITELY  DETERMINED 

ETIOLOGY. 

OF  TEMPERATE   CLIMATES. 

Acute  Articular  Rheumatism. — Various  bacteria  have  been  reported  as  cause. 

Epidemic  Poliomyelitis. — Material  from  the  cord  of  child  with  the  disease 
when  injected  subdurally,  intravascularly,  or  into  the  peritoneal  cavity  of  monkeys 
produced  the  disease  in  the  animals  inoculated.  The  virus  has  been  passed  through 
three  generations  of  monkeys  (Flexner). 

The  virus  has  been  found  in  the  brain,  spinal  cord,  mesenteric  and  salivary  glands 
of  monkeys  and  may  remain  in  the  nasal  muscosa  of  monkeys  as  long  as  five  months. 
This  would  indicate  the  existence  of  human  chronic  carriers.  With  the  possible 
exception  of  the  rabbit  only  man  and  the  monkey  are  susceptible.  This  would 
indicate  that  the  virus  is  directly  transferred  from  man  to  man.  The  virus  is 
highly  resistant  to  drying  and  light.  It  will  remain  alive  for  months  in  dust.  It  is 
not  sterilized  by  pure  glycerine  during  many  months  of  contact.  It  is  possibly 
transmitted  by  a  biting  fly,  Stomoxys  calcitrans. 

Flexner  and  Noguchi  have  recently  cultivated  the  virus  of  poliomyelitis  by 
employing  ascitic  fluid  to  which  had  been  added  a  fragment  of  sterile  rabbit  kidney 
and  nutrient  agar,  this  culture  medium  being  covered  with  a  layer  of  paraffin  oil. 
The  growth  is  obtained  under  anaerobic  conditions.  The  minute  colonies  are 
composed  of  globular  or  globoid  bodies  from  .15  to  .3  mikron  in  diameter.  These 
bodies  may  be  single  or  in  chains  or  in  masses.  In  older  cultures  bizarre  forms  are 
obtained.  Monkeys  bave  been  inoculated  with  the  cultures. 

Foot-and-mouth  Disease. — Probably  due  to  an  ultramicroscopic  organism. 

Measles. — Cause  entirely  unknown.  Hektoen  has  shown  that  blood  contains 
the  virus. 

Anderson  has  found  that  the  virus  of  measles  can  pass  through  a  Berkefeld  filter 
and  loses  its  infectivity  after  heating  for  15  minutes  at  55°  C.  In  infecting  monkeys 
it  was  found  that  the  blood  of  patients  with  measles  was  infective  only  just  before 
and  for  about  twenty-four  hours  after  the  appearance  of  the  eruption.  Mixed  nasal 
and  buccal  secretions  were  infective  for  monkeys  for  about  forty-eight  hours  from  the 
time  of  the  eruption.  The  scales  from  desquamating  cases  were  not  capable  of 
infecting  monkeys  hence  it  was  thought  that  measles  was  not  contagious  during  the 
period  of  desquamation. 

Mumps. — Herb  has  implicated  a  diplococcus.  Inoculations  into  Steno's  duct  of 
monkeys  successful. 

Rabies. — Probably  the  Negri  bodies. 

Roetheln  (German  Measles).—  Nothing  known. 

Scarlet  Fever. — Streptococci  seem  most  probable  cause  (S.  anginosus).  Mallory 
has  implicated  epithelial  protozoa. 


382  APPENDIX 

Recently  cell  inclusions  in  polymorphonuclears  have  been  supposed  to  be  diag- 
nostic; other  diseases  however  seem  to  give  them  as  measles,  diphtheria,  etc. 

Small-pox  and  Vaccinia. — Guarnieri  and  Councilman  have  implicated  epithelial 
protozoa. 

Spotted  Fever  of  the  Rocky  Mountains. — Supposed  to  be  due  to  an  unknown 
protozoon  transmitted  by  a  tick,  D.  andersoni. 

Typhus  Fever. — It  has  been  suggested  that  the  cause  may  be  a  protozoon  trans- 
mitted by  vermin. 

Recent  work  by  Anderson  and  Ricketts  has  shown  that  the  blood  of  human  cases 
is  infective  for  monkeys.  The  virus  does  not  seem  to  pass  through  a  Berkefeld 
filter  and  the  epidemiology  points  to  the  body  louse  as  the  transmitting  agent. 

Varicella. — Entirely  unknown. 

Whooping  Cough. — Influenza-like  bacilli  have  been  implicated.  Bordet- 
Gengou  bacillus. 

Or  TROPICAL  CLIMATES. 

Ainhum. — A  disease  characterized  by  a  constricting  fibrous  ring,  especially  of 
little  toe,  often  leading  to  spontaneous  amputation. 

Beriberi. — Various  microorganisms  and  food  factors  suggested.  A  form  of 
multiple  neuritis,  occurring  chiefly  in  countries  where  rice  is  the  staple  food,  char- 
acterized by  oedema  and  marked  cardiac  and  respiratory  embarrassment.  The 
vagal  involvement  produces  grave  symptoms.  Rice  from  which  the  pericarp  has 
been  largely  removed,  polished  rice,  implicated. 

Blackwater  Fever. — Considered  as  a  malarial  disease,  but  thought  by  some  to 
be  possibly  caused  by  a  protozoon — a  Babesia  (Piroplasma).  A  disease  usually 
occurring  in  malarial  patients  characterized  by  rapid  febrile  onset,  early  jaundice, 
asthenia,  pain  in  loins  and  the  pathognomonic  haemoglobinuria. 

Dengue. — Supposed  to  be  due  to  a  protozoon  transmitted  by  Culex  fatigans. 
A  disease  characterized  by  sudden  onset,  high  fever  for  three  or  four  days,  pains 
in  the  postorbital  regions,  back  and  about  joints.  A  remission  occurs  on  the  third 
to  fifth  day  followed  by  a  secondary  rise  of  temperature  and  a  measles-like  eruption. 
Leukopenia  and  reduction  in  the  percentage  of  polymorphonuclears.  Virus  exists 
in  the  blood  and  is  filterable. 

Goundou. — Symmetrical  bony  tumors  of  nasal  processes  of  superior  maxillary 
bones. 

Pellagra. — A  disease  about  which  two  etiological  views  exist  (i)  that  it  is  con- 
nected with  the  ingestion  of  spoiled  maize,  the  other  that  it  is  of  protozoal  nature 
and  transmitted  either  by  Simulium  reptans  or  Stomoxys  calcitrans.  It  is  char- 
acterized by  (i)  a  sprue-like  stomatitis  and  disorders  of  alimentary  canal  (2)  an 
erythema  usually  limited  to  parts  exposed  to  the  sun  and  characterized  by  marked 
symmetry  and  striking  delimitation  from  the  sound  skin  and  (3)  various  neuro- 
logical manifestations  and  a  toxic  psychosis  which  may  go  on  to  confusional  insanity. 
The  disease  is  characterized  by  annual  recurrences  in  the  spring  with  improvement 
in  the  winter. 

Rat-bite  Disease. — A  disease  caused  by  the  bite  of  rats.  Rather  common  in 
Japan.  Five  weeks  after  bite  when  wound  has  healed,  high  fever  sets  in,  cicatrix 


APPENDIX  383 

becomes  inflamed  with  lymphangitis  and  swollen  glands.  The  fever  falls  in  a  few 
days  to  be  succeeded  by  other  febrile  paroxysms.  An  erythematous  eruption  accom- 
panies the  second  paroxysm.  Supposed  to  be  due  to  a  protozoon. 

Sprue. — A  form  of  chronic  diarrhoea  characterized  by  diaphanous  thinning 
of  gut  and  ulcerations  of  buccal  cavity. 

Tsutsugamushi. — A  disease  of  Japan  somewhat  resembling  typhus  fever. 
Supposed  to  be  due  to  a  protozoon  transmitted  by  the  Kedani  mite. 

Verruga  Peruana. — A  disease  with  a  fever  characterized  by  a  profound  involve- 
ment of  the  bone  marrow  producing  very  rapidly  an  anaemia  resembling  that  of 
pernicious*anaemia.  Pains  of  bones  and  joints  marked.  Death  may  occur  before 
the  eruption,  which  appears  as  patients  improve,  either  as  red  spots  which  enlarge 
to  the  size  of  pea  (miliary)  or  as  pedunculated  lesions  as  large  as  a  pigeon's  egg 
(nodular).  The  lesions  are  very  haemorrhagic  and  may  appear  in  crops.  The 
nodular  form  is  located  chiefly  about  the  knees  and  elbows  but  the  miliary  form 
may  cover  entire  skin  surface  and  mucous  membranes. 

Yellow  Fever. — Supposed  to  be  due  to  a  protozoon  transmitted  by  the  Stego- 
myia  calopus.  A  disease  characterized  by  sudden  onset,  rachialgia,  albuminuria 
and  jaundice  about  the  third  day.  Pulse  becomes  slow  even  with  rising  temperature. 
Black  vomit  often  precedes  fatal  termination.  Virus  exists  in  the  blood  and  is 
filterable. 

E— CHEMICAL  EXAMINATION  OF  URINE. 

For  the  prevention  of  decomposition  when  a  urine  is  not  examined  shortly  after 
voiding,  chloroform  (10  to  20  drops  added  to  a  tightly  corked  bottle)  or  formalin 
(4  or  5  drops  to  a  pint  of  urine)  are  ordinarily  employed.  Formalin  is  better  for 
microscopical  material,  but,  owing  to  its  reducing  power,  should  be  substituted  by 
boric  acid  in  urine  to  be  examined  for  sugar.  For  clearing  urine,  turbid  by  reason 
of  bacteria,  rubbing  up  with  Talcum  purificat.  U.  S.  P.  and  filtering  is  recommended. 

A  twenty-four-hour  specimen  is  necessary  for  accurate  work.  The  urine  should 
be  collected  in  clean  separate  bottles.  Where  pus  comes  from  the  bladder  the 
proportion  of  pus  in  each  bottle  will  be  practically  the  same;  if  from  the  kidneys 
the  amount  will  vary  in  the  different  bottles. 

The  amount  of  urine  varies  in  different  individuals  (water  or  beer  habit).  It  is 
usually  given  as  from  1000  to  1500  c.c. 

Long  proposes  to  substitute  2.6  for  Haeser's  coefficient  which,  if  multiplied  by 
the  two  final  figures  of  the  specific  gravity  taken  at  25°  C.,  gives  the  weight  of  urin- 
ary solids  in  1000  c.c. 

Albumin. — Practically  serum  albumin  alone  is  clinically  important. 

The  two  usual  tests  are  i.  Heat  test  and  2.  Heller's  nitric  acid  test.  For  the 
former,  add  3  to  10  drops  of  5%  acetic  acid  to  the  perfectly  clear  urine  in  a  test- 
tube  and  bring  to  a  boil.  By  boiling  the  upper  portion  a  turbidity  in  contrast  with 
the  clear  lower  portion  may  be  obtained. 

A  more  delicate  test  for  albumin  is  the  following:  Add  to  a  test-tube  half  filled 
with  filtered  urine  one-fifth  it's  volume  of  a  saturated  aqueous  solution  of  sodium 
chloride;  heat  to  the  boiling  point;  add  two  to  five  drops  of  fifty  per  cent,  acetic 
acid  and  heat  again.  This  test  may  serve  to  distinguish  nucleo-albumin,  as  most 


384  APPENDIX 

forms  of  nucleo-proteid  found  in  urine  do  not  react  to  the  test,  while  serum  albumin 
does.  Thus  where  a  positive  nitric  acid  test  is  present,  and  no  precipitate  occurs 
with  this  test,  the  proteid  present  is  usually  nucleo-proteid. 

For  Heller's  test,  pour  a  small  amount  of  nitric  acid  into  a  narrow  test-tube 
and,  while  holding  the  tube  at  an  angle  of  about  45°,  superimpose  a  layer  of  the  urine 
to  be  tested,  which  is  delivered  drop  by  drop  from  a  pipette  and  allowed  to  flow 
down  the  side  of  the  tube. 

This  test  can  be  converted  into  a  quantitative  one  which  is  sufficiently  accurate 
for  clinical  purposes.  It  is  based  on  the  fact  that  a  specimen  of  urine  containing 
0.003%  °f  albumin  will  give  a  perceptible  ring  at  the  layering  of  the  urine  and  acid 
in  two  minutes.  If  the  ring  appears  at  once  or  in  a  few  seconds  the  albumin  con- 
tent is  greater.  From  the  qualitative  test  an  idea  can  be  formed  as  to  the  amount 
of  albumin  which  the  urine  contains,  a  heavy  ring  forming  immediately  showing  a 
considerable  albumin  content.  Probably  the  highest  elimination  of  albumin  is 
found  in  chronic  parenchymatous  nephritis  where  it  may  run  from  i  to  3%.  In 
an  ordinary  case  of  acute  nephritis  0.5%  would  be  an  average  content. 

Recently  I  have  been  using  for  both  qualitative  and  quantitative  albumin  tests 
the  apparatus  shown  in  Fig.  7.  This  is  simply  a  five-inch  piece  of  one- fourth-inch 
soft  glass  tubing  heated  at  a  point  2  inches  from  one  end,  drawn  out  for  about  two 
inches  and  bent  to  form  a  U  tube  with  one  end  shorter  than  the  other.  This  form 
of  tube  enables  one  to  perform  two  tests  with  the  same  column  of  nitric  acid  and  is 
easily  cleaned  and  dried.  They  may  be  kept  suspended  around  a  glass  tumbler's 
rim.  Taking  up  a  small  amount  of  nitric  acid  with  a  capillary  bulb  pipette  it  is 
deposited  in  the  capillary  curve  of  the  bent  tube.  This  acid  pipette  should  be 
kept  attached  to  the  acid  bottle.  With  a  second  pipette  the  urine  is  deposited  in 
the  short  arm  of  the  U  tube  and  the  presence  of  albumin  shows  by  a  distinct  ring 
at  the  junction  of  urine  and  acid  in  the  clear  capillary  tubing.  The  long  arm  will 
serve  for  the  introduction  of  a  second  specimen  of  urine  for  the  albumin  test. 

For  quantitative  test  we  dilute  the  filtered  urine  with  one  or  more  parts  of  nor- 
mal salt  solution  according  to  the  intensity  of  the  albumin  ring.  A  very  convenient 
way  of  making  the  dilution  is  with  a  graduated  centrifuge  tube.  Make  a  one  to 
ten  dilution  of  the  urine,  mix  and  draw  up  with  a  bulb  pipette  and  deposit  in  the 
short  arm  of  the  U  tube.  A  distinct  ring  forms  in  2  or  3  seconds.  Pour  off  one- 
half  of  the  diluted  urine  and  make  up  with  an  equal  amount  of  saline.  Deposit 
this  1-20  dilution  in  the  long  arm.  The  ring  forms  in  about  a  minute.  With 
further  testing  it  is  found  that  a  one  to  forty  dilution  shows  a  perceptible  ring  in 
just  2  minutes.  This  final  and  successful  dilution  multiplied  by  0.0033  gives  the 
percentage  of  albumin  in  the  urine  (40X0.0033=0.13%). 

Should  it  be  desired  to  determine  the  nature  of  the  proteids  present  either  in 
urine  or  in  exudates  or  transudates  the  following  method  is  applicable.  Determine 
the  percentage  of  total  proteid  by  the  method  employed  above.  Then  throw  down 
the  globulins  by  the  addition  of  an  equal  amount  of  a  saturated  solution  of  ammo- 
nium sulphate,  filter  and  estimate  the  proteid  content  of  the  filtrate.  The  differ- 
ence between  that  and  the  total  gives  the  percentage  of  globulin/  The  filtrate  is 
now  treated  with  5%  acetic  acid  until  a  precipitate  of  nucleo-proteid  ceases  to  form; 
the  fluid  is  filtered  and  the  clear  filtrate  (which  should  not  show  any  turbidity  with 
a  drop  of  5  %  acetic  acid)  is  tested  for  its  proteid  content,  which  represents  the 


APPENDIX  385 

serum  albumin.  When  the  combined  percentage  of  globulins  and  serum  albumin 
is  subtracted  from  the  total  proteid  percentage  we  have  the  percentage  of 
nucleo-proteid. 

Bence-Jones  Body. — (Albumose.)  Perform  the  heat  test  for  albumin.  The 
appearance  of  a  heavy  precipitate  which  partially  clears  on  boiling  suggests  albu- 
mose.  If  albumose  is  present  a  cloud  will  appear  in  the  nitrate  on  cooling.  The 
precipitate  formed  with  nitric  acid,  if  due  to  albumose,  disappears  with  heat,  that 
of  serum  albumin  does  not. 

As  another  test  for  the  Bence-Jones  body,  usually  present  in  multiple  myelomata, 
that  of  Boston  is  of  value.  Mix  15  c.c.  urine  in  a  test-tube  with  an  equal  amount  of 
saturated  NaCl  solution.  Add  2  c.c.  of  40%  NaOH  solution  and  shake  the  contents 
of  the  tube  thoroughly.  Heat  the  upper  contents  of  the  tube  to  boiling  and  add  lead 
acetate  solution  (10%)  drop  by  drop  continuing  the  heating.  A  brown  to  black 
precipitate  (sulphur)  shows  this  form  of  albumin. 

In  tests  requiring  the  removal  of  albumin  boil  the  urine  and  add  dilute  acetic 
acid  until  the  precipitate  is  flocculent,  then  filter. 

SUGAR. 

Fehling. — Pour  equal  parts  of  Fehling's  copper  solution  (34.639  grams  of  copper 
sulphate  in  500  c.c.  of  water)  and  Fehling's  alkali  solution  (173  grams  sodium  potas- 
sium tartrate  and  50  grams  sodium  hydrate  in  500  c.c.  water)  into  a  test-tube.  Mix 
and  dilute  the  deep  Wire  solution  with  two  parts  of  water.  Heat  the  upper  portion 
of  the  diluted  Fehling's  solution  in  the  flame  to  boiling  and  drop  in  from  a  pipette 
the  urine  to  be  examined.  A  yellowish  to  red  precipitate  shows  the  presence  of 
sugar. 

Fehling's  test  will  show  the  presence  of  i/ioo  of  i  %  of  glucose  in  an 
aqueous  solution  but  is  vastly  less  delicate  for  sugar  in  urine.  This  is  due  to  the 
power  of  the  creatinin  in  urine  of  holding  the  reduced  suboxide  of  copper  in  solu- 
tion. An  important  point  is  that  the  creatinin  is  broken  up  by  prolonged  boil- 
ing hence  the  puzzling  precipitates  one  gets  at  times  after  a  long  period  of  boiling 
are  explained  in  this  way.  Glycuronic  acid  may  cause  a  doubtful  reaction.  If  the 
precipitated  cuprous  oxide  is  in  very  fine  granules  the  color  is  greenish,  if  less  fine, 
greenish-yellow  and  if  quite  coarse,  reddish. 

Creatinin  holds  in  solution  the  copper  suboxide  formed  by  uric  acid  as  well  as 
that  resulting  from  very  small  glucose  content  of  urine. 

As  a  test  for  doubtful  glycosuria  it  is  well  to  give  100  grams  of  pure  glucose.  A 
normal  person  should  deal  with  such  an  amount  without  showing  sugar  reaction  of 
the  urine. 

Phenylhydrazin  (Kowarsky). — Mix  five  drops  of  pure  phenylhydrazin  in  a  test- 
tube  with  ten  drops  of  glacial  acetic  acid.  Shake  lightly  and  add  15  drops  of  satu- 
rated solution  of  NaCl.  This  makes  a  pasty  mixture.  Now  add  10  c.c.  of  the  urine 
and  bring  carefully  to  a  boil  over  a  small  flame  and  continue  to  boil  gently  for  two 
minutes.  Upon  cooling  a  yellowish  crystalline  precipitate  falls  more  or  less  rapidly 
according  to  the  sugar  content  of  the  urine.  If  the  urine  contains  0.2%  or  more  of 
sugar  the  precipitate  appears  in  a  few  minutes.  The  test  is  sensitive  for  0.03%  of 
sugar. 

25 


386  APPENDIX 

Fermentation  Test. — This  is  the  surest  test  for  sugar  in  the  urine.  It  will  show 
the  presence  of  0.05%  of  glucose.  Instead  of  the  Einhorn  apparatus  one  may  be 
extemporized  by  taking  a  50  c.c.  cylinder,  filling  it  to  overflowing  with  the  urine 
which  has  previously  been  rubbed  up  with  a  piece  of  compressed  yeast  the  size  of  a 
hazel-nut.  The  urine  should  be  made  acid  with  tartaric  acid  to  prevent  ammoniacal 
decomposition  with  the  formation  of  CO2.  A  small  3-in.  test-tube  is  filled  with  the 
yeast-treated  urine  and  dropped  mouth  downward  into  the  50  c.c.  cylinder.  The 
apparatus  is  incubated  for  twenty-four  hours  and  the  presence  of  gas  in  the  closed 
end  of  the  test-tube  shows  that  sugar  was  present.  A  control  to  determine  that  the 
yeast  does  not  contain  sugar  is  advisable.  To  utilize  this  test  as  a  quantitive  one, 
first  accurately  take  the  specific  gravity  of  the  urine;  then  add  the  yeast  and 
fill  the  test-tube  and  cylinder  as  directed  above.  Next  pour  off  or  pipette  off  the 
urine  exactly  to  the  50  c.c.  mark.  Incubate  for  twenty-four  to  forty-eight  hours 
and  make  up  the  loss  by  evaporation,  with  distilled  water.  After  the  urine  has 
cooled  down  to  room  temperature  the  contents  of  tube  and  cylinder  are  thoroughly 
mixed  (the  small  tube  having  been  withdrawn  with  a  pair  of  forceps),  then  filtered 
to  remove  the  sediment  of  yeast  and  then  brought  to  the  exact  original  volume  of 
50  c.c.  with  distilled  water  to  make  up  the  loss  by  evaporation.  (If  there  should  be 
doubt  as  to  the  completion  of  the  fermentation  of  the  glucose  a  qualitative  test  for 
sugar  can  be  made.)  The  specific  gravity  is  again  taken  and  the  difference  between 
this  and  the  first  reading  multiplied  by  0.23.  Example:  Specific  gravity  of  unfer- 
mented  urine,  1.030,  that  of  urine  after  incubation,  1.022.  Difference,  8X0.23  = 
1.84%. 

It  is  advisable  to  have  two  good  urinometers,  one  to  register  from  1000  to  1025, 
a  second  to  register  from  1025  to  1050. 

Benedict's  New  Method  for  Quantitative  Determination  of  Sugar  in  Urine. 

The  solution  for  quantitative  work  has  the  following  composition: 

Copper  sulphate  (pure  crystallized) 18.0  c.c. 

Sodium  carbonate — crystallized  (100  grams  of  anhydrous 

salt  will  answer) 200 .  o  gm. 

Sodium  or  potassium  citrate 200 .  o  gm. 

Potassium  sulphocyanate 125.0  gm. 

5  %  potassium  ferrocyanid  solution 5.0  c.c. 

Distilled  water  to  make  total  volume  of 1000.0  c.c. 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate  and  sulphocyanate  in  enough 
water  to  make  about  800  c.c.  of  the  mixture,  and  filter  if  necessary.  Dissolve  the 
copper  sulphate  separately  in  about  100  c.c.  of  water  and  pour  the  solution  slowly 
into  the  other  liquid,  with  constant  stirring.  Add  the  ferrocyanid  solution,  cool  and 
dilute  to  exactly  one  liter.  Of  the  various  constituents,  the  copper  salt  only  need  be 
weighed  with  exactness.  Twenty  five  c.c.  of  the  reagent  are  reduced  by  50  mg.  of 
glucose. 

Sugar  estimations  are  conducted  as  follows:  The  urine,  10  c.c.  of  which  should 
be  diluted  with  water  to  100  c.c.  (unless  the  sugar  content  is  believed  to  be  low),  is 
poured  into  a  50  c.c.  burette  up  to  the  zero  mark.  Twenty-five  c.c.  of  the  reagent 
are  measured  with  a  pipette  into  a  porcelain  evaporating  dish  (25-30  cm.  in  diameter), 


APPENDIX  387 

10  to  20  gm.  of  crystallized  sodium  carbonate  (or  one-half  the  weight  of  the  anhy- 
drous salt)  are  added,  together  with  a  small  quantity  of  powdered  pumice-stone  or 
talcum,  and  the  mixture  heated  to  boiling  over  a  free  flame  until  the  carbonate  has 
entirely  dissolved.  The  diluted  urine  is  now  run  in  from  the  burette,  rather  rapidly 
until  a  chalk  white  precipitate  forms,  and  the  blue  color  of  the  mixture  begins  to 
lessen  perceptibly,  after  which  the  solution  from  the  burette  must  be  run  in  a  few 
drops  at  a  time,  until  the  disappearance  of  the  last  trace  of  blue  color,  which  marks 
the  end  point.  The  solution  must  be  kept  vigorously  boiling  throughout  the  entire 
titration.  If  the  mixture  becomes  too  concentrated  during  the  process,  water  may 
be  added  from  time  to  time  to  replace  the  volume  lost  by  evaporation.  The  cal- 
culation of  the  percentage  of  sugar  in  the  original  sample  of  urine  is  very  simple. 
The  25  c.c.  of  copper  solution  are  reduced  by  exactly  50  mg.  of  glucose.  Therefore 
the  volume  run  out  of  the  burette  to  effect  the  reduction  contained  50  mg.  of  the 
sugar.  When  the  urine  is  diluted  1:10,  as  in  the  usual  titration  of  diabetic  urines, 
the  formula  for  calculating  the  per  cent,  of  sugar  is  the  following: 

'„      times  1000  equals  per  cent,  in  original  sample,  wherein  X  is  the  number  of 

cubic  centimeters  of  the  diluted  urine  required  to  reduce  25  c.c.  of  the  copper 
solution. 

In  the  use  of  this  method  chloroform  must  not  be  present  during  the  titration. 
If  used  as  a  preservative  in  the  urine  it  may  be  removed  by  boiling  a  sample  for  a 
few  minutes,  and  then  diluting  to  its  original  volume. 

This  solution  will  keep  indefinitely  and  it  is  claimed  by  Benedict,  that  compari- 
son with  the  polariscope  and  by  Allihn's  gravimetric  process  will  show  it  to  be  more 
accurate  than  any  of  the  ordinarily  used  methods. 

APPROXIMATE  QUANTITATIVE  ESTIMATION  with  Fehling's  solution.  (One  c.c.  of 
Fehling's  solution  is  reduced  by  5  mg.  glucose.) 

Measure  off  2  c.c.  of  Fehling's  solution  in  a  pipette  and  put  in  a  test-tube  or 
small  beaker  and  dilute  with  20  c.c.  of  water. 

Bring  the  diluted  Fehling's  to  boiling  and  drop  in  drop  by  drop  the  urine  from 
a  dropping-bottle  for  which  the  number  of  drops  per  c.c.  has  been  noted.  Esti- 
mating 20  drops  to  the  c.c.  if  2  drops  of  urine  are  required  to  reduce  the  copper  it 
would  show  a  sugar  percentage  of  the  urine  of  10.  Four  drops  5%,  8  drops 
2-5%>  16  drops  1.25%,  32  drops  0.6%,  64  drops  0.3%,  100  drops  0.2%. 

The  Pancreatic  Reaction  of  Cammidge  in  the  Urine. 

Cammidge  claims  that  there  is  a  definite  and  important  relationship  between 
his  pancreatic  reaction  in  the  urine  and  disease  of  the  pancreas.  The  results  of 
some  workers  go  to  support  this  view,  particularly  when  considered  in  connection 
with  the  examination  of  the  faeces  for  neutral  fat. 

The  principle  of  the  reaction  depends  upon  the  formation  in  the  urine  of  a  sub- 
stance having  the  characters  of  an  unfermentable  pentose  sugar  after  boiling  with 
hydrochloric  acid.  It  is  not  present  in  the  original  urine  as  such,  and  forms  an 
osazone  on  treatment  with  phenylhydrazine,  easily  distinguished  from  the  corre- 
sponding compound  of  glucose.  As  the  presence  of  glucose  would  seriously  interfere 
with  the  success  of  the  reaction,  all  specimens  of  the  urine  examined  must  be  care- 


388  APPENDIX 

fully  tested  for  glucose,  and,  if  present,  it  must  be  removed  by  fermentation  with 
yeast  cake.  Glucose  is  rarely  present. 

The  technic  of  the  reaction  requires  considerable  time,  but  is  easy  of  manipula- 
tion, and  should  be  readily  carried  out  in  any  hospital.  The  urine,  if  alkaline, 
must  be  made  acid  in  reaction,  and  any  albumin  or  sugar  present  must  be  removed 
and  the  urine  made  up  to  its  original  bulk  with  distilled  water.  To  40  c.c.  of  the 
clear  filtered  urine  are  added  2  c.c.  of  concentrated  hydrochloric  acid,  and  the  mix- 
ture gently  boiled  for  ten  minutes  in  a  small  flask,  using  a  funnel  in  the  neck  as  a 
condenser.  It  is  now  cooled  and  distilled  water  added  to  again  make  up  the  con- 
tents to  40  c.c.,  owing  to  the  loss  by  evaporation.  Eight  grams  of  lead  carbonate  are 
now  slowly  added  to  neutralize  the  excess  of  acid.  After  standing  for  a  few  minutes 
the  flask  is  again  thoroughly  cooled  and  the  contents  filtered  until  perfectly  clear. 
The  filtrate  is  then  well  shaken  with  8  grams  of  powdered  tribasic  lead  acetate,  and 
the  resulting  precipitate  removed  by  filtration,  which  is  repeated  until  perfectly 
clear. 

The  excess  of  lead  in  solution  must  now  be  removed  by  treating  with  4  grams  of 
powdered  sodium  sulphate;  the  mixture  is  heated  to  boiling,  then  thoroughly  cooled 
and  filtered.  From  the  filtrate  are  measured  17  c.c.;  this  is  transferred  to  a  small 
flask  with  funnel  condenser  and  there  are  added  2  grams  of  sodium  acetate,  0.8 
grams  phenylhydrazine  hydrochloride  and  i  c.c.  of  50%  acetic  acid.  The  mixture 
is  then  boiled  gently  for  ten  minutes,  filtered  into  a  test-tube  with  a  mark  showing 
15  c.c.,  and  made  up,  if  necessary,  to  that  point  with  hot  distilled  water.  The  fil- 
trate is  carefully  stirred  and  left  to  stand  over  night. 

The  quantity  and  time  of  deposit  of  the  crystals  will  depend  upon  the  degree 
of  extension  of  the  inflammatory  process  in  the  pancreas.  Thus,  in  well-marked 
cases,  a  light-yellow  flocculent  precipitate  should  appear  in  a  few  hours,  but  in  less 
characteristic  cases  it  may  be  necessary  to  leave  the  preparation  over  night  before 
a  deposit  occurs.  Under  the  microscope  the  precipitate  is  seen  to  consist  of  long, 
light-yellow,  flexible,  hair-like  crystals  of  pentosazon,  arranged  in  delicate  sheaves. 

Urinary  Tests  in  Connection  with  Acidosis. 

The  determination  of  the  ammonia  quotient,  which  is  the  ratio  of  N  eliminated 
as  ammonia  to  total  nitrogen  elimination,  has  assumed  great  importance  by  reason 
of  its  connection  with  various  forms  of  acid  intoxication,  as  in  diabetes,  pernicious 
vomiting  of  pregnancy,  and  various  hepatic  diseases. 

The  degree  of  acidosis  is  better  determined  by  the  quantitative  estimation  of 
nitrogen  elimination  as  ammonia  than  by  estimating  quantitatively  the  amount  of 
diacetic  and  /?-oxybutyric  acid  in  the  urine.  Normally  we  have  about  0.7  gram  of 
ammonia  eliminated  daily.  In  acidosis  this  may  rise  to  5  or  10  grams  and  instead 
of  being  from  3  to  5%  of  the  total  N,  it  may  amount  to  30  to  50%. 

Formalin  Method  for  the  Estimation  of  Ammonia. 

Free  ammonia  reacts  with  formalin  to  form  hexamethylenetetramine.  If 
sodium  hydrate  is  added  to  neutralized  urine  in  the  presence  of  formalin  free  am- 
monia is  liberated  and  reacts  with  the  formalin.  So  soon  as  all  the  ammonia  has 
been  liberated,  the  end  reaction  occurs. 


APPENDIX  389 

Ronchese  first  utilized  this  principle  and  Mathison  found  that  pot.  oxalate 
made  the  end  reaction  sharper.  Brown  found  that  preliminary  clearing  with  lead 
subacetate  made  the  end  reaction  still  sharper  and  removed  certain  nitrogenous 
substances  which  reacted  with  formalin  making  the  result  only  about  5%  higher 
than  with  Schaffer's  method.  The  technic  is  as  follows:  About  60  c.c.  of  filtered 
urine  are  treated  with  3  grams  of  basic  lead  acetate,  well  stirred,  allowed  to  stand 
a  few  minutes  and  filtered.  The  filtrate  is  treated  with  2  grams  of  neutral  potas- 
sium oxalate  well  stirred  and  filtered;  10  c.c. -of  the  clear  filtrate  are  diluted  to  50 
c.c.  with  distilled  water;  a  few  drops  of  i%  phenolphthalein  solution  are  added. 
The  mixture  will  be  slightly  alkaline  or  acid.  Five  grams  potassium  oxalate 
are  added  and  stirred.  It  is  exactly  neutralized  with  decinormal  NaOH  or  H2SO4. 
Twenty  c.c.  of  20%  commercial  formalin,  previously  made  neutral,  are  added,  and 
the  solution  again  titrated  with  decinormal  NaOH  to  neutralization.  Every  c.c. 
of  decinormal  NaOH  corresponds  to  0.0017  gram  NHs.  The  quantity  of  ammonia 
is  then  calculated  on  the  basis  of  the  twenty-four-hour  volume.  Example:  The 
10  c.c.  of  urine  required  4  c.c.  N/io  NaOH  to  give  a  pink  color.  4X0.0017  =0.0068. 
Then  100  c.c.  urine  would  contain  0.068  and  1000  c.c.  (twenty-four-hour  urine  am- 
ount) 0.68  gram  of  ammonia. 

ESTIMATION  OF  TOTAL  NITROGEN. 

Principle. — The  nitrogenous  material  of  the  urine  is  converted  into  ammonium 
sulphate  on  boiling  with  H2SO4.  The  ammonia  is  then  estimated  as  described 
under  estimation  of  ammonia  by  the  formalin  method. 

Technic. — Solutions  required  : 

1.  Twenty  per  cent,  commercial  formalin  previously  made  neutral  with  NaOH. 

2.  N/io  NaOH. 

3.  Forty  per  cent.  NaOH. 

Ten  c.c.  of  filtered  urine  are  pipetted  into  a  Kjeldahl  or  Koch  flask;  10  c.c.  of 
concentrated  H2SO4  and  10  grams  K2SO4  are  added.  The  mixture  is  heated  over 
a  free  flame,  gently  at  first  to  avoid  foaming,  and  is  finally  brought  to  a  boil,  which 
is  continued  until  the  mixture  is  perfectly  clear,  usually  requiring  forty-five  minutes 
to  an  hour.  The  contents  are  cooled  and  quantitatively  transferred  to  a  200  c.c. 
volumetric  flask  and  i  c.c.  of  phenolphthalein  solution  added.  The  greater  part  of 
the  acidity  is  now  neutralized  by  adding  about  30  c.c.  of  the  40%  NaOH.  It  is 
cooled  under  a  water  tap  and  made  up  to  the  200  c.c.  mark;  10  c.c.  are  taken,  diluted 
to  50  c.c.  with  distilled  water  and  exactly  neutralized  with  N/io  NaOH.  Twenty 
c.c.  of  the  formalin  solution  are  now  added  and  the  titration  again  performed.  The 
pink  end  reaction  is  beautifully  clear  and  sharp.  The  second  reading  multiplied 
by  the  factor  0.0014  gives  the  amount  of  nitrogen  in  grams  in  10  c.c.  of  the  fluid. 
It  is  then  computed  for  the  twenty-four-hour  volume  as  for  N,  eliminated  as  ammonia. 
Example:  It  required  5  c.c.  N/io  NaOH — 5X0.0014  =  0.007.  As  original  10  c.c. 
were  diluted  to  200,  the  10  c.c.  taken  for  titration  would  only  be  1/20;  hence  0.007  X 
20  =  0.14  gram  for  10  c.c  or  1.4  for  100  c.c.  or  14  grams  for  1000  c.c. 

The  amount  of  urea,  which  represents  from  85  to  90%  of  the  total  nitrogen,  is 
usually  determined  instead  of  the  total  N.  The  hypobromite  and  hypochlorite 
methods  are,  however,  lacking  in  accuracy,  and  more  exact  methods  of  urea  estima- 
tion are  more  time-consuming  than  the  one  just  given  for  total  N. 


390 


APPENDIX 


Probably  the  most  convenient  test  for  urea  is  the  hypobromite  method,  using 
the  Doremus  ureometer  with  a  side  tube  connected  to  the  closed  arm  of  the  fermenta- 
tion tube  by  a  glass  stop  cock. 

The  reagent  is  prepared  by  taking  70  c.c.  of  a  30%  stock  solution  of  NaOH, 
diluting  it  with  180  c.c.  water  and  then  adding  5  c.c.  of  bromine,  stirring  until  the 

bromine  is  dissolved.  This  solution  if  stored 
in  a  cool  dark  place  will  keep  about  one  week, 
The  urine  to  be  tested  must  be  free  from 
sugar  and  albumin  and  contain  less  than  i  % 
of  urea.  Ordinarily  the  urine  must  be  di- 
luted two  to  four  times  to  obtain  a  speci- 
men containing  less  than  i%.  In  using 
this  improved  Doremus  ureometer  the  closed 
portion  of  the  U  tube  is  filled  with  the  hy- 
pobromite solution,  and  the  urine  introduced 
by  allowing  it  to  run  in  from  the  side  tube 
by  opening  the  glass  cock  arranged  for  that 
purpose.  After  the  gas  has  risen  and  the 
instrument  has  stood  for  a  short  time  the 
readings  may  be  made  in  grams  to  the  liter, 
or  in  percentage. 

This  urea  determination  is  only  a  rough 
clinical  one. 

Gerhardt's  Test  for  Diacetic  Acid. 

Add  a  few  drops  of  ferric  chloride  solu- 
tion to  10  to  50  c.c.  of  urine  so  long  as  a 
precipitate  continues  to  form.  Then  filter 
and  to  the  filtrate  add  more  ferric  chloride 
solution.  A  bordeaux  red  color  shows  dia- 
cetic  acid.  The  test  is  sensitive.  As  a  con- 
trol to  show  that  the  color  is  not  due  to  drug 
elimination  (antipyrine,  salicylates,  etc.) 
boil  a  specimen  which  gave  the  test  for 
three  to  five  minutes.  If  the  color  was  due 

to  drugs  it  will  be  obtained  with  a  boiled  sample  while  such  treatment  drives  off  the 
diacetic  acid.  In  Hurtley's  test  add  2.5  c.c.  HC1  and  i  c.c.  of  i%  sol.  of  sod. 
nitrate  to  10  c.c.  urine.  Shake  and  allow  to  stand  2  minutes.  Now  add  15  c.c. 
strong  ammonia  followed  by  5  c.c.  of  10%  sol.  ferrous  sulphate.  The  slow  produc- 
tion of  a  violet  colour  shows  positive  test  (2  hours).  Shows  i  part  aceto-acetic 
acid  in  50,000. 

If  the  urine  shows  a  well-marked  Gerhardt  reaction  it  is  well  to  test  for  /?-oxy- 
butyric  acid. 

The  following  modification  of  Lange's  test  by  Hart  is  a  satisfactory  one.  The 
principle  involved  is  the  removal  of  acetone  and  diacetic  acid  by  heat,  then  oxidizing 
/?-oxybutyric  acid  to  acetone  with  hydrogen  peroxide  and  then  testing  for  acetone. 


FIG.   106. 


-Doremus-Hinds  Ure- 
ometer. 


APPENDIX  3QI 

Method:  Take  20  c.c.  of  urine,  dilute  with  an  equal  amount  of  water  and  add  a 
few  drops  of  acetic  acid.  Next  boil  in  a  beaker  until  the  original  amount  of  diluted 
urine  is  reduced  to  10  c.c.  (originally  40  c.c.).  Dilute  this  evaporated  urine  with  an 
equal  amount  of  water,  giving  us  20  c.c.  In  each  of  two  test-tubes  put  10  c.c.  of 
this  20  c.c.  To  one  tube  add  i  c.c.  of  hydrogen  peroxide  and  warm  gently,  without 
boiling,  for  one  minute;  then  cool.  The  other  tube  is  left  untreated.  Next,  to  each 
test-tube  add  10  drops  of  glacial  acetic  acid  and  5  to  10  drops  of  a  freshly  prepared 
sodium  nitroprusside  solution  and  mix.  Next  carefully  overlay  each  tube  with 
about  2  c.c.  of  concentrated  ammonia.  If  /2-oxybutyric  acid  were  present  in  the 
tube  treated  with  the  hydrogen  peroxide  and  thereby  oxidized  to  acetone  a  violet- 
red  ring  will  develop  at  the  point  of  contact  while  in  the  untreated  tube  there  will 
be  no  such  color  ring. 

A  yellowish-brown  ring  from  the  presence  of  creatinin  may  show  in  the  untreated 
tube.  It  is  well  to  allow  the  tubes  to  stand  for  three  to  four  hours  before  finally 
reporting  the  absence  of  /3-oxybutyric  acid.  It  will  probably  show  0.2%. 

Acetone. — To  one-sixth  of  a  test-tube  of  urine  add  a  crystal  of  sodium  nitro- 
prusside. Make  strongly  alkaline  with  NaOH.  Shake.  The  addition  of  a  few 
drops  of  glacial  acetic  gives  a  purple  color  to  the  foam,  if  acetone  is  present. 

Diazo  Reaction. — To  5  c.c.  sulphanilic  acid  solution  (sulphanilic  ac.  i  pt.,  HC1 
50  pts.,  aq.  1000  pts.)  add  two  drops  of  a  0.5%  solution  of  sodium  nitrite.  Add  an 
equal  quantity  (5  c.c.)  of  urine.  Shake  and  add  quickly  2  or  3  c.c.  of  ammonium 
hydrate.  A  carmine  color,  especially  in  the  foam,  shows  a  diazo  reaction.  If  the 
reaction  is  positive,  and  the  mixture  is  allowed  to  stand  for  24  hours,  a  precipitate 
forms,  the  upper  margin  of  which  exhibits  a  green,  greenish-black  or  violet  zone. 

Indican. — Take  10  c.c.  urine  and  treat  it  with  i  c.c.  of  sol.  of  lead  subacetate. 
Filter.  Of  this  nitrate  take  6  c.c.  and  treat  with  an  equal  amount  of  Obermayer's 
reagent;  allow  to  stand  for  5  minutes  then  shake  gently  with  2  c.c.  of  chloroform. 
Obermayer's  reagent  is  strong  HC1  containing  2  parts  of  ferric  chloride  to  the  liter 
— o.i  gram  to  50  c.c.  of  HC1. 

A  more  exact  method  is  to  pour  off  the  supernatant  acid  urine.  Wash  the 
chloroform  with  water,  then  pour  off  as  much  of  the  supernatant  water  as  possible 
and  add  10  c.c.  of  alcohol.  A  clear  blue  fluid  results. 

Urobilin. — Urobilin  appears  in  considerable  quantity  in  urine  when  there  is 
much  destruction  of  red  cells,  as  in  pernicious  anaemia,  internal  haemorrhage,  and  in 
malaria  cachexia.  The  best  test  is  that  of  Schlesinger.  To  the  unfiltered  urine 
add  an  equal  amount  of  a  saturated  solution  of  zinc  acetate  in  absolute  alcohol. 
Shake,  add  a  few  drops  of  Lugol's  solution  and  filter.  Fluorescence  in  the  nitrate 
shows  the  presence  of  urobilin.  The  degree  of  blood  destruction  is  indicated  by  the 
intensity  of  the  fluorescence. 

Bile  Pigments. — A  satisfactory  test  is  that  of  Rosin  (Trousseau).  Overlay  10 
c.c.  urine  with  about  5  c.c.  of  dilute  tincture  of  iodine  (i  to  10  of  95%  alcohol). 
An  emerald  green  ring  at  the  point  of  contact  shows  the  presence  of  bile  coloring 
matter. 

Phenolsulphonephthalein  Test  for  Renal  Efficiency. 

Geraghty  has  recently  stated  that  in  35  cases  where  an  autopsy  made  it  possible 
to  verify  the  accuracy  of  this  test  that  the  lesions  as  revealed  at  autopsy  corre- 


392  APPENDIX 

sponded  closely  with  the  results  of  the  test.  Again  in  30  nephrectomies  the  condi- 
tions found  were  in  accordance  with  the  results  of  the  test.  The  general  opinion  of 
those  who  ;have  used  the  test  is  that  it  is  more  reliable  than  cryoscopy  and  far  easier 
of  application.  The  technic  is  as  follows:  One  c.c.  of  the  phthalein  solution 
containing  6  mg.  is  injected  intramuscularly  or  subcutaneously.  The  drug  can  be 
bought  in  ampules  ready  for  use.  About  twenty  minutes  before  injecting  the  drug 
the  patient  is  given  from  200  to  400  c.c.  of  water  to  drink.  After  the  injection  the 
bladder  is  emptied  with  a  catheter  and  the  time  is  accurately  noted  when  the  urine 
which  subsequent  to  the  emptying  of  the  bladder  and  being  allowed  to  drop  into  a 
test-tube  containing  one  drop  of  a  25%  sodium  hydrate  solution  first  shows  a  pink- 
ish tinge.  This  is  recorded  as  the  time  of  appearance  of  the  drug  in  the  urine  and 
normally  is  about  10  minutes.  The  catheter  is  then  withdrawn  and  the  urine  that 
is  passed  in  the  first  hour  collected  and  subsequently  that  passed  in  the  second  hour. 
To  each  hour's  specimen  sufficient  25%  sodium  hydrate  is  added  to  give  a  purple- 
red  color  and  the  entire  amount  is  then  poured  into  a  liter  flask  and  made  up  to 
1000  c.c.  A  similar  treatment  is  employed  for  the  urine  of  the  second  hour.  The 
amount  of  drug  eliminated  in  each  hour  is  then  determined  by  a  colorimeter. 

Cabot  has  proposed  the  use  of  a  series  of  ten  test-tubes  containing  solutions  of 
the  drug  representing  from  5%  to  50%  of  the  drug  dose,  each  tube  containing  5% 
more  than  the  preceding  one.  These  comparison  solutions  may  be  made  up  with 
the  patient's  urine  obtained  at  the  time  of  emptying  the  bladder  so  that  the  con- 
fusion which  may  obtain  when  water  is  used  is  avoided.  It  has  recently  been  pro- 
posed to  make  the  standards  with  water  and  use  a  piece  of  yellow  glass  for  match- 
ing. The  urine  to  be  tested  made  up  to  1000  c.c.  as  previously  described  is  then 
poured  into  a  test-tube  of  similar  size  and  matched. 

In  normal  cases  Cabot  got  46%  of  the  drug  eliminated  in  the  first  hour,  the 
average  for  the  second  hour  being  17%.  The  quantity  of  urine  secreted  in  either 
hour  has  no  relation  to  the  test,  which  is  the  percentage  of  drug  eliminated.  In 
cases  with  serious  kidney  disease  the  amount  of  drug  eliminated  in  the  first  hour 
may  range  from  5  to  12%. 

When  the  question  of  the  kidney  involved  arises,  the  urine  must  be  taken  by 
ureteral  catheterization  or  by  a  separator. 

F— CHEMICAL  EXAMINATION  OF  GASTRIC  CONTENTS. 

The  test  breakfast  ordinarily  used  is  that  of  Ewald  (one  shredded  wheat  biscuit 
or  two  small  pieces  of  toast  with  400  c.c.  of  water  is  what  is  usually  given).  This 
Ewald  breakfast  is  a  low-grade  stimulant  to  acid  production.  It  is  given  in  the 
morning  on  an  empty  stomach.  If  at  supper,  the  night  before,  the  patient  partake 
of  raspberry  jam  the  finding  of  the  characteristic  seeds  in  the  stomach  contents 
the  next  morning  would  be  evidence  of  lack  of  motor  activity.  The  Fischer  meal 
which  contains  a  4-ounce  Hamburg  steak  in  addition  to  the  water  and  toast  of  the 
Ewald  is  withdrawn  after  three  hours. 

The  stomach  tube  is  more  easily  passed  if  it  be  thoroughly  chilled  in  ice  water 
without  the  use  of  any  lubricant. 

The  stomach  tube  should  be  passed  one  hour  after  the  Ewald  breakfast  and  if 
more  than  50  c.c.  of  fluid  be  obtained  it  indicates  stasis  or  hypersecretion. 


APPENDIX  393 

Filter  the  gastric  contents  and  test  first  for  free  HC1.  The  most  reliable  and 
sensitive  test  is  that  of  Gunsberg.  The  reagent,  which  should  be  freshly  prepared, 
consists  of  phloroglucin  3  grams,  vanillin  i  gram,  and  absolute  alcohol  30  c.c.  By 
mixing  2  drops  of  gastric  juice  and  an  equal  quantity  of  Gunsberg  reagent  in  a  small 
porcelain  dish  and  carefully  heating  above  a  flame  we  obtain  a  carmine  red  color 
if  free  HC1  be  present.  A  water  bath  is  preferable. 

For  lactic  acid  a  modification  of  Strauss'  method  is  quite  satisfactory.  Shake, 
in  a  test-tube,  5  c.c.  of  gastric  contents  with  20  c.c.  of  ether,  allow  to  settle  and 
pour  off  5  c.c.  of  the  supernatant  ether  into  another  test-tube.  To  this  ether  add 
20  c.c.  of  water  and  2  drops  of  a  i  to  9  solution  of  ferric  chloride  and  shake  well. 
The  presence  of  i%  of  lactic  acid  will  give  an  intense  greenish  color. 

Having  determined  the  presence  or  absence  of  free  hydrochloric  or  lactic  acid, 
we  should  make  a  quantitative  test  of  the  various  factors  producing  the  acidity  of 
gastric  juice  (a  modified  Topfer  test).  These  are:  i.  Free  HC1.  2.  Combined  HC1. 
3.  Acid  salts,  and  4.  Total  acidity. 

To  10  c.c.  of  filtered  gastric  contents,  in  a  beaker,  add  3  drops  of  dimethyl- 
amido-azo-benzol  solution  (a  1/2%  solution  in  95%  alcohol).  In  the  presence  of 
free  HC1  the  fluid  becomes  a  rich  carmine  pink. 

After  reading  the  burette  run  in  N/io  NaOH  solution  until  the  pink  color  is 
discharged  and  a  light  yellow  color  is  obtained.  This  reading  multiplied  by  10 
gives  the  amount  of  free  HC1  in  degrees,  a  degree  corresponding  to  i  c.c.  N/io 
NaOH.  Next  add  6  drops  of  a  1/2%  alcoholic  solution  of  phenolphthalein  to  the 
light  yellow  fluid  in  the  beaker.  Again  titrating  the  same  preparations  we  add 
N/io  NaOH  until  a  faint  but  distinct  pink  color  is  produced.  The  number  of  c.c. 
added  for  the  free  HC1  plus  the  number  to  give  the  pink  color  when  multiplied  by 
10  gives  the  total  acidity  in  degrees.  (For  example:  2.5  c.c.  N/io  NaOH  used  to 
obtain  yellow  color — 2.5X10  =  25  or  acidity  due  to  free  HC1.  After  adding  the 
phenolphthalein,  4  c.c.  N/io  NaOH  required  to  produce  pink  color — 4+2. 5  X 
10  =  65  or  total  acidity  in  terms  of  acidity.  This  means  that  it  would  require  65 
c.c.  N/io  NaOH  to  neutralize  100  c.c.  of  gastric  juice.  A  total  acidity  of  60 
is  about  normal.  To  obtain  percentage  in  HC1  multiply  by  0.00365;  thus.  65  X 
0.00365  =  0.23  HC1.) 

Having  determined  the  total  acidity  add  3  c.c.  of  10%  neutral  calcium  chloride 
solution  to  the  gastric  contents  already  in  the  beaker.  As  a  result  of  the  formation 
of  acid  calcium  phosphate  the  pink  color  is  discharged.  Again  add  N/io  NaOH 
from  the  burette  until  the  pink  color  is  restored.  The  number  of  c.c.  used  gives  the 
amount  of  acid  salts  present. 

From  the  figures  for  the  total  acidity  subtract  the  sum  of  that  for  free  HC1  and 
for  acid  salts  and  the  remainder  will  give  the  acidity  due  to  combined  HC1. 

G— CHEMICAL  TESTS  OF  FAECES. 

To  test  for  acidity  Kaplan  rubs  up  5  grams  faeces  in  30  c.c.  distilled  water.  Put 
2  c.c.  of  the  emulsion  in  a  test-tube  and  add  a  few  drops  of  phenolphthalein  solution. 
Titrate  with  N/io  NaOH  to  a  pink.  A  normal  stool  from  a  Schmidt  test-diet  re- 
quires about  1.5  c.c.  N/io  NaOH.  After  fermentation  the  stool  may  be  quite  acid 
or  more  alkaline  than  before  the  fermentation  test. 


394  APPENDIX 

Test  for  Pancreatic  Ferments. — To  obtain  a  stool  for  ferment  examination, 
calomel  2  to  3  grains,  or  phenolphthalein  5  grains  is  to  be  preferred  to  salts.  The 
Fuld-Gross-Goldschmidt  test  uses  for  trypsin  testing  a  solution  of  casein,  i  gram, 
sodium  carbonate,  i  gram  chloroform  i  c.c.  to  i  liter  of  water.  If  the  stool  is  not 
very  liquid  5  grams  of  faeces  are  rubbed  up  with  20  c.c.  salt  solution  and  filtered. 
Dilutions  of  i  to  10,  i  to  100  and  i  to  1000  are  made  and  0.5  and  i  c.c.  of  these  dilu- 
tions added  to  6  test-tubes  each  containing  5  c.c.  of  the  casein  solution.  The  tubes 
are  incubated  for  24  hours  at  38°  C.  and  completion  of  the  digestion  tested  by  add- 
ing 5%  acetic  acid  which  should  not  cause  a  precipitate  in  tubes  in  which  digestion 
is  complete. 

The  estimation  is  made  by  units,  one  unit  being  the  digestive  power  of  one  c.c. 
of  faeces  filtrate  to  digest  i  c.c.  of  casein  solution.  If  i  c.c.  of  the  i  to  1000  faeces 
dilution  digested  5  c.c.  of  casein  solution  it  would  represent  5000  units.  If  i  c.c.  of 
i  to  10  dilution  it  would  be  50.  As  there  are  5  c.c.  of  the  casein  solution  we  multi- 
ply the  dilution  of  faeces  by  5  for  i  c.c.  or  by  10  if  we  had  only  0.5  c.c.  of  faeces  dilution 
in  the  tube  tested. 

For  amylopsin  a  similar  technic  is  followed  using  a  i  %  solution  of  soluble  starch 
instead  of  the  0.1%.  casein  solution.  The  end  reaction  is  tested  by  adding  i 
drop  of  N/io  iodine  solution  to  each  of  the  starch  tubes  and  fasces  dilution  after 
24  hours  of  incubation.  The  absence  of  a  blue  color  shows  completion  of  starch 
digestion. 

The  normal  ferment  content  of  the  fasces  rarely  falls  below  200  units  and 
may  be  as  high  as  10,000.  Cases  showing  a  ferment  value  of  only  25  to  50  units 
are  very  suspicious  as  regards  pancreatic  disease. 

H— DISINFECTANTS  AND  INSECTICIDES. 

By  disinfection  is  meant  the  destruction  of  injurious  bacteria. 

Sterilization  is  where  all  living  things  are  destroyed. 

Germicides  are  substances  which  kill  bacteria  while  antiseptics  are  those  which 
are  inimical  to  the  growth  of  bacteria. 

Formalin  is  antiseptic  in  1-50,000  dilution  but  germicidal  only  in  1-20. 

Deodorants  may  or  may  not  be  antiseptic  or  germicidal.  An  insecticide  may 
or  may  not  be  a  germicide  and  vice  versa. 

In  disinfection  we  must  consider 

(1)  Strength  of  solution. 

(2)  Time  of  application. 

(3)  Nature  of  medium  in  which  disinfectant  acts. 

By  Coefficient  of  Inhibition  we  mean  time  and  concentration  necessary  to  prevent 
development  of  bacteria. 

By  Inferior  Lethal  Coefficient  we  mean  time  and  concentration  necessary  to  kill 
nonspore-bearing  bacteria. 

By  Superior  Lethal  Coefficient  we  mean  time  and  concentration  necessary  to 
kill  spore-bearing  bacteria. 

Disinfectants  may  be  (A)  physical  (B)  gaseous  (C)  chemical. 

(A)  Of  the  physical  disinfectants  we  have 

(i)    Sunlight.     The   red    and  yellow  rays    practically  inert.     The  violet   and 


APPENDIX  395 

ultra  violet  most  active.     Direct  sunlight  kills  plague  bacilli  in  less  than  one  hour 
— typhoid  bacilli  in  six. 

(2)  Burning.     Very  efficient  but  expensive. 

(3)  Boiling.     Especially  m  carbonate  of  soda  solution  for  about  one  hour  is  a 
very  efficient  disinfectant.     Nonspore-bearing  bacteria  are  killed  almost  instantly 
by  a  boiling  temperature.     One  must  remember  that  the  boiling  temperature  is 
lower  at  mountainous  elevations. 

(4)  Steam.     Extremely  efficient.     The  condensation  of  the  steam  on  the  object 
to  be  sterilized  gives  off  latent  heat  and  produces  a  vacuum. 

(B)  Of  the  gaseous  disinfectants  we  have  the  very  efficient  germicide  formal- 
dehyde gas  and  the  weakly  germicidal,  but  potent  insecticide,  sulphur  dioxide. 

Formaldehyde  gas  is  practically  valueless  as  an  insecticide. 

Bromine,  chlorine  and  hydrocyanic  acid  gas  have  a  certain  degree  of  efficiency 
but  are  not  of  practical  application.  Hydrocyanic  acid  gas  is  especially  dangerous 
on  account  of  its  extreme  toxicity. 

(i)  Formalin. — This  is  a  40  %  solution  of  formaldehyde  gas,  but  is  as  a  rule 
of  less  strength  from  evaporation  or  otherwise.  Formaldehyde  is  efficient  as  a 
surface  disinfectant  when  the  temperature  is  above  50°  F.  and  the  air  contains  at 
least  60  %  of  moisture.  It  is  not  efficient  in  cold  dry  rooms.  Owing  to  its  lack 
of  penetrating  power  it  is  not  efficient  for  the  disinfection  of  mattresses,  or  similar 
articles.  To  prepare  a  room  for  disinfection  we  must  measure  the  cubic  space 
to  ascertain  the  necessary  amount  of  formalin  to  use  and  stuff  up  or  better  paste 
up  with  newspaper  all  cracks  and  openings. 

In  the  production  of  formaldehyde  gas  the  more  expensive  autoclaves  and  lamps 
have  largely  been  replaced  by  the  simple  formalin  permanganate  method.  In  this 
one  pours  500  c.c.  of  formalin  on  250  grams  of  potassium  permanganate  for  each 
1000  cubic  feet  with  six  to  twelve  hours'  exposure. 

In  employing  this  method,  take  a  pan  partly  filled  with  water.  Place  in  this 
a  second  metal  or  glass  receptacle  containing  the  permanganate.  Then  pour  the 
formalin  on  the  permanganate  crystals..  The  gas  is  generated  in  great  amount  in 
a  few  seconds.  The  receptacle  containing  the  permanganate  and  formalin  should 
be  large  enough  to  contain  ten  times  the  volume  of  formalin,  as  there  is  a  tendency 
for  the  mixture  to  foam  over  the  sides  of  the  dish. 

Another  practical  method  is  the  formalin-sheet-spraying  one.  The  formalin 
(40%)  should  be  sprayed  on  sheets  suspended  in  the  room  in  such  a  manner  that 
the  solution  remains  in  small  drops  on  the  sheet.  Spray  not  less  than  10  ounces 
of  formalin  (40%)  for  each  1000  cubic  feet.  Used  in  this  way  a  sheet  will  hold 
about  5  ounces  without  dripping  or  the  drops  running  together.  The  room  must 
be  very  tightly  sealed  in  disinfecting  with  this  process  and  kept  closed  not  less  than 
twelve  hours.  The  method  is  limited  to  rooms  or  apartments  not  exceeding  2000 
cubic  feet.  The  formalin  may  also  be  sprayed  upon  the  walls,  floors,  and  objects 
in  the  rooms. 

Paraform  Lamps. — For  single  rooms  the  use  of  the  paraform  lamp  is  quite  con- 
venient. Special  lamps  can  be  obtained  to  burn  the  paraform  tablets  or  a  pint 
tincup  will  suffice  for  the  heating  of  i  ounce  of  paraform.  The  lamp  or  alcohol 
flame  under  the  receptacle  must  not  be  high  enough  to  ignite  the  paraform  which 
burns  readily  and  in  so  doing  does  not  give  off  formaldehyde  gas.  One  ounce  of 


396  APPENDIX 

paraform  is  sufficient  for  a  space  of  500  cubic  feet.  One  can  dissolve  2  ounces 
of  paraform  in  8  ounces  of  boiling  water  and  then  pour  this  over  4  ounces  of 
potassium  permanganate  in  a  two  gallon  pail. 

N.  Y.  Health  Department  Method.— After  a  prolonged  series  of  tests  the  N.  Y. 
Department  of  Health  gave  preference  to  the  following  formula. 

Paraformaldehyde  30  grams,  potassium  permanganate  75  grams,  water  90 
grams.  The  chemicals  are  mixed  in  a  deep  quart  pan  and  the  water  is  added  and 
the  mixture  stirred.  The  evolution  of  gas  is  slow  in  starting  but  is  complete  in 
five  to  ten  minutes. 

It  was  found  that  87%  of  the  gas  was  evolved  and  the  quantities  given  above 
suffice  to  disinfect  1000  cubic  feet  in  four  hours.  It  is  well  to  put  the  small  pan 
containing  the  chemicals  in  a  larger  one  to  prevent  danger  of  fire  and  soiling  of  the 
floor  by  the  frothing  of  the  mixture. 

Sulphur  Dioxide. — Sulphur  dioxide  is  fairly  efficient,  but  requires  the  presence 
of  moisture.  It  is  only  a  surface  disinfectant  and  is  lacking  in  penetrating  proper- 
ties. An  atmosphere  containing  4.5%.  can  be  obtained  by  burning  5  pounds 
of  sulphur  per  1000  cubic  feet  of  space.  This  amount  requires  the  evaporation  or 
volatilization  of  about  i  pint  of  water.  Under  these  conditions  the  time  of  ex- 
posure should  be  not  less  than  twenty-four  hours  for  bacterial  infections.  A  shorter 
time  will  suffice  for  fumigation  necessary  to  kill  mosquitoes  and  other  vermin.  Dry 
sulphur  dioxide  produced  by  burning  2  pounds  of  sulphur  for  each  1000  cubic  feet 
of  space  will  answer  for  this  purpose.  An  exposure  of  from  two  to  three  hours  is 
sufficient. 

The  sulphur  may  be  burned  in  shallow  iron  pots  (Dutch  ovens),  containing  not 
more  than  30  pounds  of  sulphur  for  each  pot,  and  the  pots  should  stand  in  vessels 
of  water.  The  sulphur  pots  should  be  elevated  from  the  bottom  of  the  compart- 
ment to  be  disinfected  in  order  to  obtain  the  maximum  possible  percentage  of  com- 
bustion of  sulphur.  The  sulphur  should  be  in  a  state  of  fine  division,  and  ignition 
is  best  accomplished  with  alcohol  (special  care  being  taken  with  this  method  to 
prevent  damage  to  cargo  or  vessel  by  fire),  or  the  sulphur  may  be  burned  in  a  special 
furnace,  the  sulphur  dioxide  being  distributed  by  a  power  fan.  This  method  is 
peculiarly  applicable  to  cargo  vessels. 

Liquefied  sulphur  dioxide  may  be  used  for  disinfection  in  place  of  sulphur  dioxide 
generated  as  above,  it  being  borne  in  mind  that  this  process  will  require  2  pounds 
of  the  liquefied  gas  for  each  pound  of  sulphur,  as  indicated  in  the  above 
paragraphs. 

Sulphur  dioxide  is  especially  applicable  to  the  holds  of  vessels  or  to  apartments 
that  may  be  tightly  closed  and  that  do  not  contain  objects  that  would  be  injured 
by  gas.  Sulphur  dioxide  bleaches  fabrics  or  materials  dyed  with  vegetable  or  ani- 
line dyes.  It  destroys  linen  or  cotton  goods  by  rotting  the  fiber  through  the  agency 
of  the  acids  formed.  It  injures  most  metals.  It  is  promptly  destructive  of  all 
forms  of  animal  life.  This  property  renders  it  a  valuable  agent  for  the  extermina- 
tion of  rats,  insects,  and  other  vermin.  Sulphur  dioxide  is  a  germicide  only  in  the 
presence  of  moisture,  and  even  then  will  not  kill  spore-bearing  organisms.  If  cloth- 
ing is  washed  immediately  after  sulphur  disinfection  the  rotting  effect  will  be  greatly 
lessened.  If  used  in  spaces  containing  machinery  all  metal  parts  should  be  coated 
with  vaseline. 


APPENDIX  397 

CHEMICAL  SOLUTIONS. 

Bichloride  of  mercury  is  usually  sold  in  the  form  of  antiseptic  tablets.  As  a 
disinfectant  for  the  infectious  diseases  it  is  usually  used  in  a  strength  of  i-iooo. 
The  solution  should  be  made  in  a  wooden  or  earthenware  vessel.  As  bichloride 
forms  inert  albuminates  it  should  not  be  used  in  the  disinfection  of  sputum,  faeces 
or  any  albuminous  excreta.  It  must  be  remembered  that  bichloride  is  a  mordant 
so  that  any  stains  in  soiled  clothing  will  remain  permanent.  For  disinfection  of 
clothing  the  material  should  be  left  in  i-iooo  bichloride  for  one  hour.  Dishes 
for  food  should  never  be  disinfected  in  bichloride  on  account  of  the  danger  from 
poisoning.  Floors  and  walls  may  be  disinfected  with  i-iooo  bichloride  applied 
with  a  mop.  Allow  the  solution  to  dry  on  the  floor  or  walls. 

Formalin. — A  5%  solution  of  commercial  formalin  in  water  (50  c.c.  formalin 
950  c.c.  water)  makes  a  satisfactory  disinfectant  for  soiled  clothing.  It  is  also 
valuable  for  albuminous  material.  The  disinfectant  must  act  in  a  strength  of  5% 
so  that  if  one  pint  of  faeces  is  to  be  disinfected  we  should  add  one  pint  of  a  10%  for- 
malin solution  and  allow  it  to  act  for  one  hour. 

Carbolic  Acid. — It  is  soluble  in  water  to  the  extent  of  about  5%  and  in  such 
strength  it  is  an  efficient  disinfectant.  The  solution  should  be  made  with  hot  water. 

In  standardizing  disinfectants  carbolic  acid  is  used  as  the  standard.  It  how- 
ever is  expensive  and  there  is  often  difficulty  in  making  up  satisfactory  solutions. 
More  efficient  and  more  convenient  is  the  Liquor  cresolis  comp.  U.  S.  P.  This  may 
be  prepared  by  mixing  up  equal  parts  of  cresol  and  soft  soap  as  noted  on  page  12. 
This  has  a  value  according  to  tests  made  in  the  Hygenic  Laboratory  of  3,  making 
it  in  tests  without  organic  matter  three  times  as  efficient  as  carbolic  acid.  Under 
similar  conditions  lysol  had  a  value  of  2.12  creolin  3.25  and  trikresol  of  2.62. 

Equal  parts  of  a  5%  solution  of  Liq.  Cresol.  Comp.  and  the  faeces,  urine 
or  sputum  to  be  disinfected  is  satisfactory  for  disinfection  provided  the  mixture 
is  allowed  to  stand  for  one  hour.  Liq.  Cresol.  Comp.  (5%)  is  an  excellent 
disinfectant  for  contaminated  bedclothing,  etc.  It  is  also  most  suitable  for  the 
disinfection  of  floors  and  walls. 

Lime. — It  must  be  remembered  that  air-slaked  lime  is  inert  as  a  disinfectant. 
For  disinfecting  faeces  freshly  prepared  milk  of  lime  is  excellent.  It  is  made  by 
mixing  unslaked  lime  with  four  times  its  volume  of  water.  An  equal  quantity 
should  be  added  to  the  faeces  to  be  disinfected. 

Chlorinated  Lime. — This  can  be  purchased  in  air-tight  containers  and  when  the 
package  is  opened  it  should  give  off  a  powerful  odor  of  chlorine. 

For  a  working  disinfectant  solution  add  i  pound  to  4  gallons  of  water. 
This  is  satisfactory  for  mopping  floors  and  for  disinfecting  faeces,  sputum  and  urine, 
equal  parts  of  the  excreta  and  disinfecting  solution  being  mixed  and  allowed  to  stand 
for  one  hour.  For  disinfection  of  drinking  water  one  teaspoonful  of  Jchlorinated 
lime  to  i  pint  of  water  makes  a  stock  disinfectant.  For  use  one  teaspoonful  of 
this  stock  solution  is  added  to  2  gallons  of  the  drinking  water  to  be  disinfected. 
Let  stand  at  least  1/2  hour. 

INSECTICIDES. 

The  following  notes  are  taken  chiefly  from  the  U.  S.  P.  H.  Service  directions. 
SULPHUR  DIOXIDE — obtained  as  described  above — destroys  all  animal  life. 


398  APPENDIX 

In  the  case  of  vessels,  when  treated  for  yellow  fever  infection,  the  process  shall 
be  a  simultaneous  fumigation  with  sulphur  dioxide,  2  %  volume  gas,  and  two 
hours'  exposure,  in  order  to  insure  the  destruction  of  mosquitoes. 

In  the  case  of  vessels  when  treated  for  plague  the  process  with  sulphur  dioxide 
shall  be  as  follows: 

Without  cargo:  The  simultaneous  fumigation  with  sulphur  dioxide  gas  not  less 
than  2%  for  six  hours'  exposure. 

With  cargo:  Fumigation  with  sulphur  dioxide  gas,  4  %,  six  to  twelve 
hours'  exposure,  according  to  stowing. 

Infected  vessels  may  require  partial  or  complete  discharge  of  cargo,  and  frac- 
tional fumigation  for  efficient  deratization. 

Pyrethrum.  The  fumes  of  burning  pyrethrum  may  be  used  to  destroy  mos- 
quitoes in  places  where  there  are  articles  liable  to  be  injured  by  the  use  of  sulphur. 

Four  pounds  per  1000  cubic  feet  space  for  two  hours'  exposure  will  kill,  all  or 
practically  all  of  the  mosquitoes  but  precautions  should  be  taken  to  sweep  up  and 
destroy  any  that  may  have  escaped. 
Pyrethrum  stains  walls,  paper,  etc. 

The  oxides  of  carbon,  as  used  at  Hamburg,  are  efficient  to  destroy  rats  but  do 
not  kill  fleas  or  other  insects.  They  are  obtained  by  burning  carbon,  coke,  or  char- 
coal, in  special  apparatus,  and  the  gas  as  produced  consists  of  about  5  % 
carbon  monoxide,  18  %  carbon  dioxide,  and  77  %  nitrogen. 

Twenty  kilos  of  carbon,  coke,  or  charcoal  are  used  for  every  1000  meters  of 
space.  The  gas  is  allowed  to  remain  in  the  ship  for  two  hours  and  from  seven  to 
eight  hours  are  allowed  for  it  to  leave  it.  This  is  about  equivalent  to  i  1/3  pounds 
of  carbon  (coke)  to  1000  cubic  feet  of  air  space.  As  this  gas  is  very  fatal  to  man 
and  gives  no  warning  of  its  presence,  being  odorless,  a  small  amount  of  sulphur 
dioxide  should  be  added  to  give  warning  of  its  presence.  As  it  does  not  kill  fleas 
it  cannot  be  depended  on  for  complete  work,  where  there  is  evidence  of  plague 
among  rats  on  the  vessel,  as  the  infected  fleas  would  infect  the  rats  coming  aboard 
after  the  deratization. 

The  articles  named  as  disinfectants  which  can  obviously  destroy  animal  life  can 
be  used  for  that  purpose  when  applicable,  as  steam  for  bedding,  fabrics,  etc.  For- 
maldehyde is  not  applicable  for  this  purpose. 

For  fleas  the  best  insecticides  are  (i)  crude  petroleum  (fuel  oil)  which  is  at  times 
called  Pesterine,  (2)  an  emulsion  of  kerosene  oil  made  as  follows:  kerosene  20  parts, 
soft  soap  i  part  and  water  5  parts.  The  soap  is  dissolved  in  the  water  by  aid  of 
heat  and  the  kerosene  oil  gradually  stirred  into  the  hot  mixture. 

For  cockroaches  there  is  nothing  so  good  as  sodium  fluoride.  By  sprinkling 
the  powder  about  the  haunts  of  the  cockroaches  they  are  gotten  rid  of  in  a  few  days. 

For  exterminating  rats  and  in  this  way  secondarily  the  rat-fleas  besides  the 
ordinary  poisons  such  as  As.,  P.,  etc.  Rucker  has  recommended  a  poison  composed 
of  plaster  of  Paris,  6  parts,  pulverized  sugar  i  part  and  flour  2  parts.  This  mixture 
should  be  exposed  in  a  dry  place  in  open  dishes.  To  attract  the  rats  the  edge  of 
the  dish  may  be  smeared  with  the  oil  in  which  sardines  have  been  packed. 

Wise  and  Minett  report  good  results  from  the  use  of  crude  carbolic  acid  as  a 
larvicide  for  mosquitoes.  They  added  about  i  teaspoonful  for  each  2  cubic  feet 
of  water  in  the  pool.  Of  course  the  ordinary  method  for  destroying  mosquito  larvae 
is  by  covering  the  surface  of  the  water  in  the  cistern  or  pool  with  a  layer  of  petroleum. 


INDEX 


Abbe  condenser,  4 
Abscess,  bacteria  in,  356 
Acanthia  lectularia,  291,  294 

rotundata,  294 
Acarina,  282 
Acidosis,  388 
Acartomyia,  316 
Acetone,  for  sections  (see  tissue),  372 

in  urine,  391 
Acid-fast  bacteria,  76,  77 

siaining,  35 
Acid  proofing,  u 
Actinomycosis  (see    Discomyces),    123, 

336, 356 
.Kdina?,  314 
Agar,  egg,  23 

gelatin,  North,  24 

glucose,  22 

glycerine,  23 

nutrient,  21 

placenta],  30 

plating,  41 
Ancylostoma  duoderiale,  263,  274,  350, 

358 
Agglutination,  macroscopical,  144 

microscopical,  143 
Ainhum,  382 

Air,  bacteriological  examination  of,  135 
Albumin  in  urine,  383 
Albumin  in  sputum,  336 
Albumose  in  urine,  385 
Aldridr'a,  314 
Alexin,  140 
Amboceptor,  139,  151 
Ammonia  in  urine,  388 
Amoebae,  219,  336,  348,  356 
Anaemia,  aplastic,  190 

infantum,  205,  229 

pernicious,  190,  200 

primary,  199 

secondary,  201 
Ana-robes,  64,  67 

Buchner  method,  68 

combination  method,  69 

cultivation  of,  67 

Liborius  method,  68 

Tiroz/i's  method,  68 

Yignal  method,  69 

Wright  method,  69 


Anaphylaxis,  161 
Anaphylactic  shock,  162 
Anginas,  331 
Anguillula,  264 

Animal  inoculations,  48,  143,  152 
Animal  parasites,  general  classification, 
211 

mounting  of,  377 

nomenclature  in,  213 

preservation  of,  378 
Anisocytosis,  189 
Anophelinae,  291,  314 
Anthrax,  63,  64 

vaccination,  64 
Antiformin,  334 
Antigen,  138,  147,  151 
Antitoxin,  138 

botulism,  70 

diphtheria,  87 

pyocyaneus,  no 

tetanus,  73 
Antivenins,  320 
Appendicitis  blood  count,  197 
Arachnoidea,  281 
Argas,  281,  287 
Arneth  index,  193 
Ascaris,  canis,  263,  278 

lumbricoides,  263,  277,  332 
Ascitic  fluid  (cytodiagnosis  in),  360 
Aspergillus,  concentricus,  117,  123 

flavus,  123 

fumigatus,  123 

nidulans,  123 

pictor,  123 

repens,  123 

Auchmeromyia  luteola,  303 
Azolitmin,  24 

Babesia,  242 

Bacillus,  acidi  lactici,  133 

acidophilus,  108 

acnes,  357 

aerogenes  capsulat.,  64,  75,  349 

Aertyrck,  104 

anthracis,  64 

anthracis  symptomat.,  64 

bifidus,  108 

botulinus,  70 

bulgaricus,  108 


399 


4OO 


INDEX 


Bacillus,  cloacae,  108 

coli,  107, 130,  338,  356 

diphtherias,  77,  85,  328,  330 

dysenteriae  105,  349 

enteritidis  (Gartner),  70,  104 

enteritidis  sporogenes,  127 

fecalis  alkaligines,  99 

fusiformis,  331 

icteroides,  100 

influenzas,  92 

lactis  agrogenes,  107 

leprae,  82,  326,  328,  358 

mallei,  84,  328,  358 

mycoides,  63 

of  avian  tuberculosis,  79 

of  Bordet-Gengou,  91,  94 

of  bovine  tuberculosis,  79 

of  chancroid,  94 

of  chicken  cholera,  96 

of  Hofmann,  89 

of  hog  cholera,  104 

of  Koch-Weeks,  93,  326 

of  malignant  cedema,  69 

of  mouse  septicaemia,  95 

of  Morax,  94,  326 

of  smegma,  76,  82 

of  timothy  grass,  77 

of  trachoma  (Muller),  91 

paratyphosus  (A.  and  B.),  104,  352 

pestis,  95,  295,  335,  352,  355 

pneumoniae  (Friedlander),  95 

prodigiosus,  no 

proteus,  105 

pseudotuberculosis  rodentium,  92 

psittacosis,  92 

pyocyaneus,  109 

subtilis,  63 

suipestifer,  104 

tetani,  72,  356 

termo,  339 

tuberculosis,  78,  326,  328,  334,  349 

typhosus,  100,  131,  196,  349,  352 

violaceus,  109 

vulgatus,  63 

xerosis,  89,  325 

zopfii,  92 

Bactera,  identification  of,  41 
Balantidium  coli,  231 
Band's  disease,  204 
Bed  bug,  in  Kala  azar,  230 
Bence-Jones  albumin,  385 
Benedict  sugar  test,  386 
Beriberi,  382 
Bienstock  group,  92 
Bile  media,  26,  351 
Bile  pigments,  391 
Bilharziasis,  251 

infection  in,  339 


Binucleata,  217 
Black  water  fever,  382 
Blood,  coagulation  rate,  185 

color  index  of,  188 

counting  red  cells,  174 

counting  white  cells,  176 

counting  with  microscopic  field,  177 

cultures  of,  351 

differential  count  (normal),  194 

differential  count  (in  haemacytom- 
eter),  178 

dried  films,  179 

fixation  of,  181 

fresh  preparations,  177 

making  preparations,  171 

normal  count,  199 

occult,  1 86 

red  cells  of,  188 

specific  gravity  of,  186 

spectroscopic  test,  187 

staining  of,  1 79 

tubercle  bacilli  in,  352  ^ 

viscosity  of,  185 

white  cells  of,  190 
Blood  platelets,  195 
Blood  serum,  coagulating  apparatus,  10 

preparation  of,  25 
Boas-Oppler  bacillus,  354 
Booker  group,  92 

Bordet  and  Gengou  phenomenon,  146 
Bordet  amd  Gengou  bacillus,  94 
Bothriocephalus,  258 
Bottle  bacillus,  357 
Botulism,  70 
Bouillon,  glycerine,  21 

calcium  carbonate,  21 

Liebig's  extract  in,  20 

nutrient,  17 

standardizing  reaction  of,  18 

sterilization  of,  20 

sugar,  20 

sugar-free,  20 
Broth  media,  17 
Buccal  secretions,  330 

Calliphora  vomitoria,  303 

Cammidge  reaction,  350,  387 

Capsule  staining,  37 

Carbol-fuchsin  stain,  33,  35,  160 

Casts  in  urine,  343 

Cellia,  314 

Cells,  in  blood,  188,  190 

in  cytodiagnosis,  360 
Cerebrospinal  fluid,  60,  147,  360 

puncture  for.  360 
Cestoda,  245,  253 

key  to  genera,  255 
Charcot-Leyden  crystals,  333,  350 


INDEX 


401 


Chlorinated  lime,  397 
Chlorosis,  199 
Cholera,  112 

carriers  in,  114 

diagnosis,  115 

in  water,  132 

media  for,  28 
Cholera  red,  21,  116 
Chironomidae,  305 
Chlamydozoa,  243 
Chromatin  stains,  40,  183 
Chromidia,  217 
Chromogens,  109 
Chrysomyia  macellaria,  303 
Chrysops,  299 
Chyluria,  267 
Cladorchis  watsoni,  249 
Cladothrix,  117 
Classification,  animal  kingdom,  211 

arachnoidea,  281 

bacilli,  branching,  76 

bacilli,  gram  negative,  91 

bacilli,  spore  bearing,  63 

bacteria,  43 

cocci,  49 

flat  worms,  245 

fungi,  117 

insects,  291 

mosquitoes,  291,  313 

protozoa,  216 

round  worms,  263 

spirilla,  112 
Clonorchis  endemicus,  248 

sinensis,  248 
Coccidiaria,  233 
Coccidium  (see  Eimeria  and  Isospora), 

233 

Coley's  fluid,  in 
Colon  bacillus,  107 

in  water,  130 
Colonies,  isolation  of,  43 
Color  index,  188 
Colubrine  snakes,  319 
Commensalism,  213 
Complement,  140 

absorption  of,  146 

deviation  of,  145 
Conjunctival  infections,  325 
Conorhinus,  294 
Conradi-Drigalski  medium,  28 
Conradi-brilliant  green  medium,  28 
Corrosive  sublimate,  397 
Cover-glasses,  2 
Cover-glass  preparations,  32 
Crithidia,  230 
Cryptococcus  gilchristi,  120 

linguae  pilosae,  120 
Ctenopsylla  musculi,  296 
26 


Culicinae,  315 

Culture  media,  agar,  21 

bile  media,  26 

blood  agar,  26 

blood  serum,  25 

bouillon,  17 

cholera  media,  28 

egg  media,  23,  25 

faeces  media,  27 

gelatin,  23 

gelatin  agar  (North),  24 

Hiss'  serum  water,  21 

litmus  milk,  24 

peptone  solution,  21 

potato,  25 

protozoal,  29 

Russell's  double  sugar,  29 

sterilization  of,  "5,  16 

sugar  bouillon,  20 

titration  of,  18 
Cycloleppteron,  314 
Cysticercus,  255 
Cystitis,  344 
Cytodiagnosis,  359 
Cytorrhyctes  luis,  244 

scarlatinae,  244 

vaccinae,  244,  364 

Dark  ground  illumination,  4,  224 

Davainea  madagascariensis,  258 

Demodex  folliculorum,  284 

Deneke's  spirillum,  112 

Dengue,  382 

Dermacentor  andersoni,  289 

Dermatpbia  cyaniventris,  304 

Desk-microscopic,  n 

Dhobies  itch,  124 

Diazo  reaction,  391 

Dibothriocephalus  latus,  258 

Dicroccelium  lanceatum,  247 

Dieudonne's  cholera  medium,  28 

Differential  leukocyte  count,  194 

Diphtheria,  85 

diagnosis  of,  88,  331 
diphtheria-like  bacilli,  89 
media  for  growing,  25 
Neisser's  stain,  36,  88 
toxin  of,  87 

Diplococcus,  crassus,  57 

intracellular.  meningitidis,  59 
lanceolat.,  55 

Diplognoporus  grandis,  259 

Diptera,  298 

Dipylidium  caninum,  215,  228 

Disinfecting  solution,  12,  394 

Disinfectants,  394 

Distomiasis,  247 

Dorset's  egg  medium,  26 


402 


INDEX 


Double  boiler,  n 
Dracunculus,  265 
Dum  dum  fever,  229 
Dunham's  solution,  21 
Dysentery,  amoebae  in,  220,  348 

bacilli,  105 

bacilli  in  fasces,  349 

Ear  affections,  328 
Eberth  group,  99 
Echinococcus  cysts,  259 
Echinorhynchus  gigas,  279 
Ehrlich,  blood  film  method,  180 

granule  staining,  190 

tri-acid  stain,  182 
Eimeria  stiedae,  233 
Emery's  test  147 
Ekiri,  107 
Endo  medium,  27 
Endomyces  albicans,  120 
Endothelial  cell  in  cytodiagnosis,  360 
Entamceba,  buccalis,  222 

coli,  219 

histolytica,  220,  348 

tetragena,  221 
Eosinophiles,  193 
Eosinophilia,  196 
Escherich  group,  99 
Eustrongylus  gigas,  273,  339 
Exudates,  359 
Eye-piece  (see  Ocular),  2 
Eye-strain,  4 
Eye  infections,  325 

Faeces,  345 

amoebae  in,  348 

bile  in,  348 

culturing,  349 

diet  for  examination  of,  346 

fats  in,  347 

fermentation  test,  348 

pancreatic  test,  394 

plating  media,  27 

soaps  in,  347 
Fasciola  gigantea,  251 

hepatica,  247 
Fascioletta  ilocana,  250 
Fasciolopsis  buski,  249 
Fat  in  faeces,  347 
Fauces,  330 
Favus,  122 

Fehling  sugar  test,  385 
Fermentation  tubes,  10 
Films  (blood),  179 
Filter  pump,  13 
Filterable  viruses,  365 
Filaria,  bancrofti,  267,  339,  358 

demarquayi,  268 


Filaria  embryos,  key  to,  269 

loa,  267,  327,  358 

medinensis,  265 

ozzardi,  269 

perstans,  268 

philippinensis,  269 

powelli,  269 

volvulus,  268 
Fixation,  blood  films,  181 

tissues,  371 
Flagella  staining,  38 
Flagellata,  222 
Flat  worms,  245 
Fleas,  295 

key  to,  296 

Flugge's  droplet  infection,  99,  136 
Flukes,  245 

of  blood,  251 

of  intestines,  249 

of  liver,  247 

of  lungs,  250 
Focus,   microscopical,  3 
Foot  and  mouth  disease,  381 
Formalin,  395 
Friedlander  group,  91,  95 
Frozen  sections,  377 
Fungi,  Achorion,  122 

Ascomycetes,  119 

Aspergillus,  123,  327 

classification  of,  117 

Cryptococcus,  120 

cultivation  of,  125 

diagnosis  of,  125 

Discomyces  bovis,  123 

Discomyces  madurae,  124 

Hyphomycetes,  123 

Imperfecti,  123 

Madurella  mycetomi,  124 

Malassezia  furfur,  124 

Microsporoides,  124 

Microsporum  audouini,  122,  327 

Mucor,  118 

Penicillium,  123 

Rhizopus,  118 

Saccharomycetes,  119 

Trichophyton,  121 

Trichosporum  giganteum,  124 

Gall  stones,  345 
Gartner  group,  100 
Gas  production,  47 
Gastric  contents,  354 

chemical  examination  of,  392 
Gastrodiscus  hominis,  249 
Gelatin,  2,  3 

liquefaction  of,  46 

General  paralysis  (spinal  fluid  in),  361 
Gentian  violet  stains,  33 


INDEX 


403 


Giemsa's  stain,  183 
Glanders,  84,  358 
Glassware,  cleaning  of,  8 
Glossina  palpalis,  302 
Gnathostoma  siamense,  264,  358 
Gonococcus,  27,  57,  326,  339 
Gonorrhoea,  57 
Goundou,  382 
Grabhamia,  316 
Gram  method,  33 

negative  bacteria,  34 

positive  bacteria,  34 

solution,  34 

Granular  degeneration  (red  cells),  189 
Granules  (white  cells),  191 
Guinea  worm,  265 

Haemacytometer,  174 
Haemadipsa  ceylonica,  280 
Haematopota,  299 
Haematoxylin  stain,  184 
Haemin  crystals,  186 
Haemoglobin  estimation,  172 
Haemoglobinometers,  Miescher's,  172 

Sahli's,  172 

Tallquist,  173 
Haemosporidia,  235 
Haffkine,  cholera  vaccine,  115 

plague  prophylactic,  99 
Halzoun,  247 
Hanging  drop,  9 
Hemokonia,  195 
Heredity,  214 
Herpetomonas,  230 
Heterogenesis,  214,  264 
Heterophyes  heterophyes,  249 
Hirudo,  medicinalis,  280 

nilotica,  280 
Hiss'  serum- water,  21 
Histoplasma,  230 
Hodgkin's  disease,  203 
Hook  worms,  274,  350 
Hosts,  214 

Hydatid  disease,  259,  356 
Hydrocele  agar,  26 
Hymenolepis,  nana,  257 

diminuta,  258 
Hyphomycetes,  123 
Hypoderma  diana,  304 

Illumination,  dark  ground,  4 
Immersion  objectives,  3 
Immune  sera,  antimicrobic,  139 

antitoxic,  139 

diphtheria,  87 

in  diagnosis,  141 

preparation,  141 

tetanus,  73 


Immunity,  active,  138 

natural,  137 

passive,  138 
Incubators,  body  temperature,  12 

electrical,  12 

petroleum  lamp,  12 

room  temperature,  13 
Indican  in  urine,  391 
Indol,  test  for,  21 
Influenza,  92 
Infusoria,  231 
Inoculation  animals  (tuberculosis),  48, 

77 

animals  (plague),  99 

of  media,  47 
Insecticides,  397 
lodophilia,  185 
Insecta,  292 
Isospora  bigemina,  235 
Itch  mite,  283,  358 
Ixodidae,  285 

Japanese  river  fever,  283,  383 
Joints,  gonococcus  in,  59 

Kaiserling  solution,  379 

Kala  azar,  229 

Kedani  mite,  283,  383 

Key  to  branching,  curving  bacilli,  76 

to  cocci,  49 

to  filarial  embryos,  269 

to  fleas,  296 

to  Gram-negative  bacilli,  91 

to  spirilla,  112 

to  spore-bearing  bacilli,  63 
Kidney  diseases,  table,  344 
Koch's  postulates,  47 
Kundrats  lymphosarcoma,  204 

Laboratory  desks,  n 
Lactic-acid  bacteria,  108 
Lactophenol,  378 
Lamblia  intestinalis,  231 
Lamp,  primus,  15 
Larvae,  fly,  303 

mosquito,  309 

mounting,  278 
Leeches,  280 
Leishmania,  229 

media  for,  30 

donovani,  229 

infantum,  229 

tropica,  229 
Leprosy,  82,^326,  358 

diagnosis  of,  83 

in  rats,  83 
Leptothrix,  117 
Leukaemia,  202 


404 


INDEX 


Leukaemia,  lymphatic,  203 

splenomyelogenous,  202 
Leukocytosis,  197 
Leukopenia,  196 
Levaditi  stain,  375 
Leydenia  gemmipara,  222 
Light  in  microscopical  work,  4 
Linguatula  rhinaria,  289 
Liquefaction  of  gelatine,  46 
Litmus,  24 
Liver  abscess,  198 
Loeffler  serum,  25 
Loemopsylla  cheopis,  296 
Luetin,  226 
Lumbar  puncture,  360 
Lymphocytosis,  199 
Lymphocytes,  large,  191 

small,  191 
Lymphosarcoma,  204 

Madura  foot,  124 
Macrogamete,  237 
Magnifying  power,  171 

of  oculars,  2 
Malaria,  235 

cultivation,  242 

diagnosis  of,  242 

differential  tables,  240,  241 

life  cycle,  235 

life  history,  235 

index,  239 

Romanowsky  stain  in,  183 
Mallein,  85 

Mallory's  amoeba  stain,  40 
Malta  fever,  61 
Mansonia,  316 
Marchi  method,  376 
Mast  cells,  193 
Measles,  381 
Megarhininae,  314 
Meat  poisoning,  70,  104 

group  of  bacteria,  104 

toxin  of,  105 
Malignant  pustule,  64 
Mechanical  stage,  i 
Media  (see  culture  media),  16 
Megaloblast,  189 
Melaniferous  leukocytes,  199 
Meningococcus,  59,  331 
Metorchis  truncatus,  249 
Micrococcus,  53 

catarrhalis,  61 

cinereus,  50 

melitensis,  61,  339 

pharyngis  siccus,  50 

rheumaticus,  53 

tetragenus,  54 
Microgametocyte  237 


Micrometer  disk,  2,  169 

standardization  of,  1 70 

screw,  3 

Micrometry,  2,  169 
Microscope,  i 
Microscopical  sections  (see  tissue),  373 

quick  diagnostic  method,  373 
Milk,  bacteriological     examination     of, 
132 

B.  bulgaricus  in,  133 

lactic-acid  bacteria  in,  133 

leukocytes  in,  134 
Mites,  282 

Mononuclear  leukocytes,  172 
Mosquitoes,  anatomy  of,  307 

classification  of,  313 

dissection  of,  311 

larvae  of,  309 

ova  of,  309 

pupae  of,  310 
Motility  43,  45 

Brownian,  43 

current,  43 

Moulds  (see  Fungi),  117 
Mounting  parasites,  377 
Much's  granules,  35,  82 
Mucidus,  316 
Mumps,  381 
Mus  norvegicus,  296 
Musca  domestica,  300 
Muscidae,  300 
Mutualism,  212 
Myeloblasts,  195 
Myelocytes,  174 
Myzomyia,  314 
Myzorhynchus,  314 

Nasal  infections,  diphtheria  in,  328 

leprosy  in,  328 
Nastin  in  leprosy,  83 
Necator  americanus,  276 
Negri  bodies,  362 
Neisser's  stain,  36,  331 
Nematocera,  298,  305 
Nematoda,  263 
Neosporidia,  233 
Nervous  tissue,  376 
Nissl  method,  376 
Nitrogen  determination,  388,  389 
Nocardia,  125 
Noguchi  test,  153 

media  for  treponemata,  30 
Normal  solutions,  380 
Nomenclature,  in  animal  parasitology, 
213 

law  of  priority  in,  213 
Normoblasts,  189 
North's  gelatin  agar,  24 


INDEX 


405 


Notes,  blank,  bacteriology,  165 
blood  work,  206 
parasitology,  animal,  321 

Novy  MacNeal  (N.N.N.)  medium,  29 

Numerical  aperture,  3 

Nyssorhynchus,  314 

Occult  blood,  1 86 
Ocular  infections,  325 

animal  parasites  in,  327 

bacilli  in,  325 

gonococcus  in,  326 

M.  catarrhalis  in,  61 

pneumococcus  in,  326 
Objectives,  i 
Oculars,  2 

CEsophagostoma  brumpti,  273 
(Estridse,  304 
Opisthorchis,  felineus,  249 

noverca,  249 

sinensis,  248 
Opsonic  power,  156 

apparatus  in,  13 

determination  of,  157 
Ornithodoros,  287 
Orthorrhapha,  298 
Otitis,  329 

Ova  in  faeces,  253,  261,  277 
Oxyuris  vermicularis,  279 

Pancreatic  tests,  350,  394 
Pangonia,  299 
Panoptic  staining,  40 
Paragonimus  westermani,  250 
Parasitism,  242 
Parthenogenesis,  214 
Pasteurized  milk,  134 
Pasteurelloses,  95 
Pebrine,  233 

Pediculoides  ventricosus,  284 
Pediculus  capitis,  292 

vestimenti,  292 
Pellagra,  67,  382 
Penicillium  crustaceum,  123 
Petri  dishes,  41,  42 
Phagocytosis,  156 
Pleiffer's  phenomenon,  115 
Pharyngeal  secretions,  330 
Phenolsulphonephthalein  test,  391 
Phenylhydrazin  test,  328 
Phlebotomus,  306 
Phthirius  pubis,  293 
Physaloptera,  273 
Piedra,  124 
Pinta,  123 
Pipettes,  bacteriological,  14 

capillary  bulb,  15 
Piroplasmata,  242 


Plague,  95 

diagnosis  of,  99 

flea  in,  98,  295 

pneumonia,  98 

prophylaxis,  99 
Platinum  wire,  12 
Pleural  fluids  (cytodiagnosis),  359 
Pneumococcus,  55,  326 
Poikilocytes,  189 
Poliomyelitis,  381 

Polymorphonuclear  leukocytes,  193 
Porocephalus  constrictus,  289 
Protista,  217 
Protozoa,  216 

culture  of,  29 

discussion  of,  217 

staining  of,  39 
Pseudoleukaemia,  203 
Psychodidae,  306 
Pulex,  cheopis,  296 

irritans,  296 
Pulicidae,  295 
Pupipara,  299 
Pus,  cultures  from,  355 

tetanus  in,  74 
Pyretophorus,  314 

Rabies,  362 

preservation  of  dog  in,  364 
Rats,  296 

Rat-bite  disease,  382 
Reaction  of  media,  18,  45 

standardization  of,  18 
Red  blood-cells,  counting  of,  1 74 

fformal,  189 

nucleated  red  cells,  189 

polychromatophilia,  189 

punctate  basophilia,  189 
Relapsing  fever,  223 
Rhabditis  pellio,  264 
Rheumatism  (acute),  381 
Rhinosporidium,  243 
Rhizoglyphus  parasiticus,  383 
Rhizopoda,  218 
Rhizopus,  119 
Rhynchota,  293 
Rice  cooker,  n,  16 
Ring- worms,  121 

Rocky  Mountain  spotted  fever,  289,  382 
Roetheln,  381 
Romanowsky  stains,  183 
RosS  thick  film,  181 
Round  worms,  263 
Row's  haemoglobin  medium,  30 
Russell's  double  sugar  medium,  29 

Sabouraud's  medium  for  moulds,  125 
Saccharomyces,  anginosae,  119 


406 


INDEX 


Saccharomyces,  blanchardi,  120 

cerevisiae,  119 
Sarcina  lutea,  53 
Sarcoptes  scabiei,  283 
Sarcophaga  carnaria,  304 
Sarcopsylla  penetrans,  297 
Sarcosporidia,  243 
Scarlet  fever,  381 
Schistosomum  hsematobium,  251 

japonicum,  252 

mansoni,  251 
Schizotrypanum,  228 
Screw  worm,  303 

Sections,  making  and  staining,  3  73 
Septicaemia,  55 

Serum  (see  immune  serum),  141 
Sewage,  iri  water,  127 
Shiga's  bacillus,  105 
Simulidae,  305 
Siphonaptera,  295 
Siphunculata,  291,  292 
Skin  infections,  357 

itch  mite,  358 

leprosy  in,  358 

pus  cocci  in,  357 

sarcopsylla,  in,  358 
Sleeping  sickness,  227,  302 
Slides,  cleaning,  8 

concave,  9 
Smallpox,  244,  365 
Snakes,  317 
Sparganum,  mansoni,  262 

prolifer,  262 
Spectroscope,  187 
Spirillum  choleras  asiaticae,  112 

metschnikovi,  112 

of  Finkler,  Prior,  112 

tyrogenum,  112 
Spirochaeta,  222 

duttoni,  223,  287 

recurrentis,  223,  293 

refringens,  224 

vincenti,  224 
Spiroschaudinnia,  223 
Splenic  anaemia,  205 
Splenomegaly,  204 
Spores,  spore-bearing  bacilli,  63 

staining,  39 
Sporotrichosis,  124 
Sporotrichum  f eurmanni,  1 24 
Sporozoa,  233 
Sprue,  383 
Sputum,  333 

albumin  test  in,  336 

amoebae  in,  336 

antiformin  for,  334 

centrifugalization  for  T.B.,  334 

culturing,  335 


Sputum,  fixing  smears,  333 

Paragonimus  eggs  in,  336 

plague  pneumonia,  335 
Stage,  warm,  4 
Staining  methods,  32 
Stains,  acid  fast,  35 

agar  jelly,  39 

Archibald's,  36 

Balch's,  183 

capsule,  37 

carmine  for  worms,  278 

carbol  fuchsin,  33 

flagella,  38 

for  Negri  bodies,  363 

Giemsa's,  183 

Gram's  method,  33 

haematoxylin,  184,  376 

Herman's,  35 

Leishman's,  183 

Levaditi's,  375 

Loffler's  methylene  blue,  33 

Neisser's,  36 

Nicolle's,  36 

Panoptic,  40 

Pappenheim's,  36 

Ponder's  diphtheria,  37 

protozoal,  40 

Romanowsky,  36,  183,  375 

Smith's  formal  fuchsin,  35 

spore,  39 

tri-acid,  182 

Van  Giesen's,  375 

Wright's,  182 
Staphylococcus,  50,  54 

epidermidis  albus,  54 

pyogenes  albus,  54 

pyogenes  aureus,  54 
Stegomyia,  315 
Sterilization,  Arnold,  5,  16 

autoclave,  5,  16 

glass  ware,  5 

hot  air,  5 

pathogenic  bacteria,  8 
Stomach  contents,  354,  392 

Boas-Oppler  bacillus,  354 

cancer  cells  in,  354 
Stomoxys,  301 
Stool  examination,  345 
Streptococcus,  49,  50 

capsulatus,  56 

coli  gracilis,  49 

fecalis,  51 

pyogenes,  52 
Streptothrix,  125 
Strong,  cholera  prophylactic,  115 

plague  vaccine,  99 
Strongylidae,  272 
Strongyloides  stercoralis,  264 


Remarks 

Found  in  faeces  and  sewage  -contami- 
nated water.  Differs  from  B.  typhos. 
by  marked  alkali  production. 

Blood  cultures  first  week  —  'agglutina- 
tion afterward. 

Nonacid  strain,  highly  toxic. 

Acid  mannite  strain,  moderate  toxici* 

Much  like  Flexner  strain.  No  ac  id 
maltose. 

Found  in  summer  diarrhoea  of  childn 

Little  gas.  No  fluorescence  n.  rt 
Litmus  milk  acid  in  third  day. 

Much  gas.  Marked  reduction  n. 
with  yellow  fluorescence.  Litr. 
milk  alkaline  third  day.  2. 

B.  choleras  suis,  B.  icteroides,  1. 
Danysz  virus  and  B.  paratypL 
closely  related  (Gaertner  group 

There  is  also  a  B.  coli  anaerogenes  which 
is  like  B.  coli  but  does  not  form  gas 

Very  nearly  related  to  Friedlander's  bac- 
illus as  well  as  to  B.  coli.  3. 

Differs  from  B.  coli  in  liquefaction  of 
gelatin  and  shows  slow  production 
of  gas  in  lactose. 

Three  types  —  Proteus  vulgaris  rapid 
gelatin  liq.;  P.  mirabilis,  slow  gelatin 
liq.;  P.  zenkeri,  no  gelatin  liq.  Spread- 
ing growths  characteristic.  2. 

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